JPS6057837B2 - Method for producing cephalosporin compounds - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a process for the enzymatic production of cephalosporin compounds from cephalosporin C homologs.

Prior Art Pelkey Patent No. 736934, British Patent No. 12727
No. 69, JP-A-47-39595, JP-A-50-10
No. 1584 discloses the use of D-amino acid oxidase derived from Aspergillus, Penicillium, Neurospora, Aerobacter or Trigonopsis variabilis under aerobic conditions.

British Patent No. 127276 discloses a method for producing 3-acetoxymethyl-(4'-carboxybutanamide)cefu-3-emu-4-carboxylic acid by adding sodium azide. Problems to be Solved by the Invention Although enzymatic oxidative deamination of cephalosporin c homologues is important for developing and producing cephalosporin derivatives having excellent antibacterial activity, there have been few success stories.

In particular, in the case of cephalosporin c, in which D-α-aminoadipic acid is bonded with an acid amide bond, there is a problem that ordinary D-amino acid oxidase is difficult to act on it. Furthermore, simply enzymatic oxidation of cephalosporin c homologues with D-amino acid oxidase results in the formation of 3-acetoxymethyl-7-(5'-carboxy5''-oxopentanamide)cefu-3-emu-4-carboxylic acid derivatives and 3- Although an acetoxymethyl-7-(4″-carboxybutanamide)cefu-3-emu-4-carboxylic acid derivative can be obtained, it is not possible to selectively produce the derivative, which is industrially disadvantageous, and is still insufficient. do not have. Means for Solving the Problem The inventors have devised a 7-
The advantageous intermediate 3-acetoxymethyl-7-(5-carboxy5'') for producing aminocephalosporanic acids
-Oxopentanamide) Cef 3-Emu 4-carboxylic acid or 3-acetoxymethyl-7-(4''-monocarboxybutanamide) Cef 3-Emu 4-carboxylic acid was isolated from the searched microorganism in soil, Gk -340
I learned that it is generated by n.

Furthermore, as a result of intensive research, the present inventors discovered that a small amount of hydrogen peroxide (35%) was added during the contact between the enzyme and the compound of general formula 1.
By adding 0.1 to 0.5% of
-Oxopentanamide)Sefu 3-Emu 4-carboxylic acid production is completely suppressed, and 3-acetoxymethyl-7-(4''-carboxybutanamide)Sefu 3-Emu 4-carboxylic acid is produced without reducing the yield. The present invention has been completed based on the discovery that it is possible to easily and selectively isolate even the following.

In other words, the present invention provides a method for adding a compound represented by the formula C (wherein R represents a hydrogen atom, a hydroxyl group, an acetoxyl group, or a nucleophilic residue) or a salt thereof contained in a cephalosporin C fermentation process to a fermentation process. Filamentous fungus G that has the ability to directly produce D-amino acid oxidase without further extraction.
After contacting the culture of K-34 1st Microtechnical Research Institute No. 3276 with aeration in an aqueous medium, catalase was added in the presence of sodium azide and hydrogen peroxide solution. This is a method for selectively producing a cephalosporin compound represented by the formula (wherein R represents the same group as above) or a salt thereof by inhibiting the activity.

The characteristics of culturing the above-mentioned filamentous fungus Gkml ■ strain on various media are as described below.

A: Macroscopic observation 1 Zavesk agar medium Growth is rather fast, reaching a diameter of 3 Tfa in 3 days.

The hyphae are initially white, but as conidia are formed, they turn olive yellow (011veYe110w).
When cultured for an even longer period of time, the color becomes bright yellowish green (CObaltG).
reen).

Conidial formation is very common. The surface of the village is fluffy. The back side has a light edge color (0pa11neGreen) to reddish purple (Mag
enta).

No diffusible dye is produced. 2 Growth on wort agar medium was slower than on Zawbek agar medium as hyphae elongation was suppressed and grew to 2wn in diameter in 3 days.

The color is white at the initial stage of culture, but becomes grass green as conidia are formed. Conidia are formed early and are very numerous. The surface of the village is velvety. The back side is light yellow (Cream) to reddish purple (Magenta). No diffusible dye is produced. 3 Growth on oatmeal agar medium is fast and expandable, 41 cells in 3 days! Reach Rm.

The hyphae are initially white, but as conidia form, they turn dark green (F
Orest Green) and water droplets are formed on the surface. There are many conidia. The surface of the village is velvety. The back side is light yellow (P
aleYellOw). B. Microscopic Observation The conidiophores are asymmetrically branched and have a length of 30 to 70 μm and a width of 1.9 μm at the base, but the walls are generally 3 μm wide and smooth and somewhat thick.

No basal branches (Branchesraml), basal basidia (Hetulae) 7 x 0.7μ connected to conidiophores
branches out. On top of that, pen-tip-shaped firerides grow in clusters,
Conidia each having a circular shape of 1.5 μ to 1.8 μ×21 μ are linked together. The number of conidial chains is about 1 cap on oatmeal agar, about 1 cap on Zavesk agar, and about 20 to 20 on wort agar.
There are 3 pitfalls. These chained conidia grow densely and form large fan-shaped agglomerates. No cleistocysts or sclerotia are observed. C
Growth conditions Growth temperature is 15-40°C, growth pH is 3.0-7. Optimum growth temperature is 28-377C1 Optimum growth pH is 4.0-
It is 5.0.

The taxonomic position of the filamentous fungus Gk-340 strain having the above mycological properties was determined by J.C. Gilman''SA Manua.
A Manual of the Penicilia (1OfSOilFungi), Raper, Tom (co-authored)
fTllePeniciIlla) and Parnett's Imperfect Juange (111ustrated
Ger1era0fImperfectfur1gi)
I, which is considered to be a close relative of this strain,
As a result of comparative study with FO-6617, this strain has a penicillium-like appearance to the naked eye, but its conidial structure was observed under a microscope, and it was found to be a member of the genus Gliocladium (GIiOcladium).
um).

This strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as No. 3276.

In culturing enzyme-producing bacterial strains, in addition to glucose and starch as carbon sources, peanut flour and cottonseed meal as nitrogen sources, potassium phosphate and magnesium sulfate as inorganic salts, D-α-
D to induce aminoadipic acid side chain oxidase
- Amino acids are required. When D-glutamic acid is used, it has been found that an addition amount of 0.1 to 3% significantly affects oxidase production, and that an increase in the addition amount is proportional to the production amount. DL
-Glutamic acid is also effective, but it is desirable to contain 0.3% or more of D-glutamic acid. 28℃
Culture with aeration for approximately 2 m.

A significant amount of oxidase-containing microbial cells accumulated through such induction culture can be used even after being filtered, but the microbial cells should be further frozen and thawed or the live microbial cells should be brought into contact with Alucard C etc. (
Enzyme activation can be further promoted by incubating at 0.1% solution for 1 hour). When applying and reacting with Cephalosporin C culture process, in order to prevent waste of D-α-aminoadipic acid side chain oxidase, the filtrate of Cephalosporin C culture process is mixed with a weakly acidic cation exchange resin such as Amperlite IRC-50 (Na+). Pretreatment (passed as an acidic liquid with pH 6.0) is preferable.

6 to 10% of GK-34 cells were added to this pretreatment solution, and the pH was adjusted to 7. After adjusting the temperature to 0~7.5, the reaction temperature was 33'Cl
l~5v. v. m. ventilation under 10r. p. m. The reaction is allowed to proceed for 2 to 3 hours with the above high-speed stirring. When an enzymatic reaction is performed by combining the above activation technique and pretreatment with an ion exchange resin, the desired product can be obtained in high yield from the cephalosporin C culture process without any enzyme inhibition.
In fact, as mentioned above, when D-amino acid oxidase-activated bacterial cells are brought into contact with the compound of general formula 1, two compounds, namely 3-acetoxymethyl-7-(5''-carboxy-5''-oxopentanamide) are produced. Cefu 3-emu 4-carboxylic acid and 3-acetoxymethyl-7-(
4-carboxybutanamide) cef 3-emu 4-carboxylic acid is produced. At this time, if sodium azide and hydrogen peroxide are used to inhibit catalase activity,
3-acetoxymethyl-7-(4'-carboxybutanamido)cef-3-emu-4-carboxylic acid is selectively produced. When producing 3-emu-4-carboxylic acid, 0.1 to 0.2% during the oxidation reaction.
of sodium azide was added, and after 3 minutes during the reaction, 35
Add 0.25% hydrogen peroxide solution, and after 6 powders add 0.1% hydrogen peroxide solution.
Add 5%.

The amount of 3-acetoxymethyl-7-(4″-carboxybutanamide)cefu-3-emu-4-carboxylic acid produced can be determined by measuring its W absorption (260r1m) after extraction with an organic solvent such as ethyl acetate. Quantify.

At the same time, completion of the reaction is also confirmed by thin layer chromatography (developing solvent: isopropanol/water = 7:3).
After the oxidation reaction, the pH of the reaction solution is lowered to 6.0 or less and filtered.
The reaction filtrate can be used as it is or concentrated to have a pH of 1.5 to 2.
．． After adjusting to 0, extraction is performed with an organic solvent that is immiscible with water, such as methyl isobutyl ketone, n-butanol, ethyl acetate, etc. If necessary, the reaction solution may be subjected to the following concentration operation. That is, the reaction filtrate was treated with 15% of the strongly basic anion exchange resin (for example, PA-30-1RA-4).
01 etc.) and adsorb it to an acetic acid type of resin (1.5~2).
After washing with twice the amount of water, 0.2 to 1. Elute with CM ammonium chloride solution. The eluate was concentrated under reduced pressure, and 1 of the reaction solution was
110 to 1112. This concentrate is PHL. 5～
2.1 and extracted three times with the same amount of an organic solvent that is immiscible with water, and the organic solvent was distilled off under reduced pressure to obtain 3-
A crude powder of acetoxymethyl-7-(4″-carboxybutanamide)cefu-3-emu-4-carboxylic acid is obtained.

Adding salt such as common salt during solvent extraction is one of the preferred methods for completely performing solvent extraction. To purify the crude powder, add 3 times the volume of methanol to completely dissolve it, and then add 30 to 40 f8 amounts of ether to precipitate impurities.

When the ether layer is concentrated after filtration, needle-shaped crystals are precipitated, so after the ether layer is filtered off, it is washed again with chloroform and dried under reduced pressure. Alternatively, the sodium salt can be precipitated by dissolving the crude powder in an equal amount of methyl isobutyl ketone and adding sodium-2-ethylhexanoate.

3-acetoxymethyl-7-(4″-carboxybutanamide)cefu-3-emu-4-carboxylic acid extracted from the reaction solution with an organic solvent was simply concentrated to 113 to 114 without distilling off the entire amount of the organic solvent. It is also possible to add dicyclohexylamine and precipitate and separate the respective salts.

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

Example 1 Gliocladium GK-34 Yui was mixed with 4% joint flour, 1.5% peanut meal, and 0.0% monobasic potassium phosphate.
After inoculating 60 ml of a preculture liquid medium containing 2% magnesium sulfate, 0.05% magnesium sulfate, and culturing with shaking at 28°C for 24 to 48 hours, it was incubated with glucose 4%, peanut meal 1.5%, D
- Transfer to 250 ml of sheet medium containing 0.5% glutamic acid, 0.2% monobasic phosphate, and 0.05% magnesium sulfate, and culture with shaking at 28'C.

This sheet 1e contains 4% glucose and 2% peanut meal.
, DL-glutamic acid 0.5%, monopotassium phosphate 0.1%
, tank medium 130 containing 0.05% magnesium sulfate
1 and cultured at 2 g'C with aeration for about n hours, but the culture was stopped when the pH of the prosthesis rose to 5.0. At this time, 130 ml of Argard C was added and after stirring for 1 hour, the mixture was filtered and washed with water using a filter breath to obtain 8k9 wet bacterial cells with activated D-amino acid oxidase. This is -20℃
It was stored frozen. On the other hand, the cephalosporin C culture process was filtered, and the filtrate 601 (containing 216' as cephalosporin C) was diluted with 2N-NaOH to pH 6. Corrected to O, Amperlite 1RC-50 (SV = 3
Let it pass.

Immediately correct the pH of the passed-through liquid to 5.5 to 6.0. When carrying out the oxidation reaction, use this IRC-50 treatment liquid 60e (205.2V as cephalosporin C).
After adjusting the pH to 2N-NaOH'C-PH7.5 and bringing the temperature to 33'C in a tank equipped with an aeration stirring device, 90f of sodium azide was added, and 5k9 of the live wet bacterial cells were added. 3v. v. m. under ventilation of 200r. p. m
．． Stir with.

After the reaction starts, the pH decreases until about the first 2 minutes, so P
H7. It was maintained at O~&0. After the third powder, add 150 ml of 35% hydrogen peroxide solution, and after the sixth powder, add 100 ml.

Qualitative thin layer chromatography detection (developing solvent is isopropanol/water = 7:3) is performed every 30 minutes during the reaction. In addition, 1 ml of sample was taken every hour and the pH was 2. O, add 8 ml of ethyl acetate, shake for 5 minutes, separate 5 ml from the ethyl acetate layer, and add 5 ml of 0.4M NaHCO3 solution.
After further shaking for 5 minutes, the absorption of the aqueous layer at 260r1m was measured, and the amount of 3-acetoxymethyl-7-(4''-carboxybutanamide)cefu-3-emu-4-carboxylic acid produced was measured.The results , 1400 after 1 hour of reaction
r'/ml, which reached 2500 r/ml in the second hour.
The oxidation reaction was completed in about 2 hours, and the reaction solution had a pH of 6. After adjusting to O, 1 kg of filter aid was added and filtered using a filter breath to obtain filtrate 7.8e.

When this filtrate is measured using high-speed liquid chromatography,
It was estimated that 146y of 3-acetoxymethyl-7-(4''-carboxybutanamide)cefu-3-emu-4-carboxylic acid was produced.The reaction filtrate was diluted to pH 6.
Amperlite IRA-401 (CH3CO
O-)91SV=3 and washed with 13.5e of water. At that time, unreacted cephalosporin C47.8y was measured in the adsorption waste liquid and washing liquid by the hydroxylanomine method. The aforementioned IRA-401 resin is 0.2M-
NH4Cl4.5elO. 5M-NH4Cll3.5e
．． ．． The active fraction 18e was obtained by stepwise elution with IM-NH, Cl25e.

A portion of this active fraction was collected, extracted with ethyl acetate, and the sodium bicarbonate inversion solution had an absorption measurement value of 102 f at 260 r1m. This active fraction was azeotroped with n-butanol 5e and concentrated to 5e, then added with 1.5k9 of common salt and purified with concentrated hydrochloric acid.
Hl. 8, and then 3 with methyl isobutyl ketone 51'.
Extracted twice.

This extract was completely distilled under reduced pressure to obtain a crude powder of 93V of 3-acetoxymethyl-7-(4''-carboxybutanamide)cefu-3-emu-4-carboxylic acid.Example 2 To purify the crude powder of 3-acetoxymethyl-7-(4″-carboxybutanamide)cefu 3-emu-4-carboxylic acid prepared by the same method as in 1, add 360 ml of methanol to 120 y of the above crude powder and dissolve it. Afterwards, 3.6e of ether was further added, the resulting precipitate was filtered, and the filtrate was concentrated under reduced pressure to 100 ml to yield needle-like crystals.

After filtering the crystals and washing with 200 ml of chloroform,
Drying under reduced pressure gave crystals 82y of 3-acetoxymethyl-7-(4''-carboxybutanamide)cefu-3-emu-4-carboxylic acid.The elemental analysis value of this was Cl5H.
8N2O8S was as follows. Example 3 Inhibitors were removed from the culture filtrate of Cephalosporin C using Amperlite IRC-50 (Na+) in the same manner as in Example 1.
containing V) and D-amino acid oxidase activated bacterial cells 50
0y and sodium azide 9y are reacted at 33° C. for 2 hours under ventilation.

During the reaction time, 15 ml of 35% hydrogen peroxide solution was added after 3 minutes from the start of the reaction, and 10 ml was added after 6 minutes.
The amount of the target product produced was a measured value of 12.4y. This reaction-completed solution was added with 30% sulfuric acid to PHL. 8 and filtered to obtain filtrate 6.3e.

The filtrate was cooled to 5° C. and extracted three times with an equal volume of methyl isobutyl ketone to collect the solvent layer 6.3e. Concentrate this to 1.96e, add 0.6y of activated carbon, and
After stirring, filter.

This filtrate and 100 m of methyl isobutyl ketone
Collect the filtrate after washing with activated carbon using 1. 7.8 ml of dicyclohexylamine was added to this methyl isobutyl ketone solution, stirred at 10°C, and the precipitate was filtered. This precipitate was first washed with 900 ml of methyl isobutyl ketone, then washed again with acetone 1e and dried to form 3-acetoxymethyl-7-(4''-carboxybutanamide)cefu 3-emu 4-carboxylic acid. 16.1y of the dicyclohexylamine salt was obtained.

Example 4 20y of crude powder of 3-acetoxy methyl-7-(4″-carboxybutanamide)cefu 3-emu-4-carboxylic acid prepared in the same manner as in Example 1 was added to 200 m
Dissolve 1 in ethyl acetate and filter to remove insoluble materials.
After concentrating to 13 to 11 [sodium-2-ethylhexanoate (4). 05M) 7.6y/ethyl acetate 2
2 ml] of the solution was added with stirring to precipitate a yellow sodium salt.

This was washed twice with ethyl acetate (approximately 30 ml) and dried to obtain sodium salt crystals 16y of 3-acetoxymethyl-7-(4''-carboxybutanamide)cefu-3-emu-4-carboxylic acid.Reference Example 1 Dry bacterial cell powder 4 having D-amino acid oxidase activity
5fI was added to Cephalosporin C Pros 1.5' pretreated with Amperlite IRC-50Na, and 700 m9 of sodium azide was added to adjust the pH to 8. The reaction was carried out at 33° C. with stirring at an air flow rate of 2 V′/v per minute.

The reaction was completed in 5 hours and 3-acetoxymethyl (5″
-carboxy5″-oxopentanamide) Cefu 3
-Emu-4-carboxylic acid reached a certain amount one hour after the start of the reaction and did not increase any further. Finally, 26.2% of cephalosporin C was converted to 3-acetoxymethyl-(5''-carboxy5''-oxopentanamide)cefu-3-emu-4-carboxylic acid, and the equivalent of 54.2% was converted to 3- It was converted to acetoxymethyl-7-(4'-carboxybutanamide)cefu-3-emu-4-carboxylic acid. Effects of the Invention According to the production method of the present invention, a culture of a microorganism belonging to the genus GliOcladium, which has the ability to produce D-amino acid oxidase in a cephalosporin c homolog, or a treated product thereof is aerated in an aqueous medium. 3-aminocephalosporanic acid derivatives are useful intermediates for 7-aminocephalosporanic acid derivatives by contacting them with
Acetoxymethyl-7-(4'-monocarboxybutanamide)cefu-3-emu-4-carboxylic acid derivatives can be easily obtained selectively.

Claims (1)

[Claims] A compound represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼ (I) (wherein R represents a hydrogen atom, a hydroxyl group, an acetoxyl group, or a nucleophilic residue) or Without extracting the salt from the fermentation broth, a culture of a microorganism belonging to the genus Gliocladium that has the ability to produce D-amino acid oxidase or a processed product thereof is aerated in an aqueous medium. Formulas that are characterized by inhibiting catalase activity in the presence of sodium azide and hydrogen peroxide after contact ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) (R is the same group as above) A method for producing a cephalosporin compound or a salt thereof represented by