JPS59210897A - Production of beta-lactam compound - Google Patents

Abstract

NEW MATERIAL:A beta-lactam antibiotic TAN-458 represented by formula I and its salt. USE:It shows antibacterial activity against gram-positive and -negative bacteria and is used as a synthetic intermediate of medicines and as a reagent. PREPARATION:A microorganism in Spirillum, capable of producing antibiotic TAN-458 such as Spirillum sp-UK-1521 strain (FERM-7075) is cultured aerobically at about 10-35 deg.C and a pH of about 4-8 and the objective antibiotic TAN- 458 is obtained mainly from the filtrate of the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a novel antibiotic TA, pr-458, and its salts.

In order to search for new antibiotics, researchers isolated a large number of microorganisms from the soil and searched for all the antibiotics they produced. The microorganism belongs to the genus Spirillum, and by culturing the microorganism in an appropriate medium, an antibiotic that exhibits antibacterial activity against Gram-positive bacteria and Durum-negative bacteria is added to the medium. After learning about the accumulation of scales, we isolated this antibiotic and confirmed from its physicochemical and biological properties that it was a β-lactam antibiotic represented by the following formula.

The first compound has already been synthesized by D. Cama et al. (J. Amer. Chem. Soc.
, 100 + 8006 (+978)), the compound (
Because the sodium salt) is unstable, its physicochemical properties are not clearly described. In addition, W, L, Parker et al. named this compound (free carboxylic acid) as 5Q27860, and after derivatizing it to p-nitrobenzyl ester, it was converted to Serasia (
Serratia) M and Erwi
nia) has been isolated from cultured gI as a substitute product of FA bacteria (J, Antibiotics, 35.
653 (1982)).

In this case as well, it is impossible to isolate compound (I) from the culture medium, and therefore its physicochemical properties are not described.

The present inventors recognized these findings and completed the present invention as a result of further research.

That is, the present invention provides a method for producing a compound of the above formula, which is characterized by culturing in a medium a bacterium that produces a compound of the formula ( Learn about methods for producing compounds or their salts.

In addition, in this application, the antibiotic T A xq -458 may be simply referred to as T A N -458.

Antibiotic γ′f T AN-458 used in the present invention
Spirillum (Spirillum) is used as a raw sake.
1) Any species may be used as long as it belongs to the genus and has the ability to produce the antibiotic TAN-458.

As an example of antibiotic TAN-458-producing bacteria, UK 1521 strain of the genus Spirillum (Spirillum') collected from soil in Hyogo Prefecture by the present inventors is used.
Hereinafter, it may be abbreviated as "rUK-1521 strain". )
can be given. The mycological properties of UK-1521 strain of the genus Nubilium are as follows.

(a) Morphology Observation after culturing for 2 days on a broth agar slant at 2El showed that the necrocysts were curved or spiral-shaped with a width of 0.6-0.9μ and a length of 2.5-12μ, with pleomorphism. Not shown.

It is mobile and has a polar flagellum. does not form spores;
In addition, Guhum staining was negative and showed no acid-fast properties.

(b) Growth status on various media The cells were cultured at 28° C. and cultured in one inkstone for 1 to 14 days.

■ Meat'tf basic plate culture = After 3 days of culture, a colony of round, single-headed, full-light lilies with a diameter of 0.1 to 0.5 silk is formed.

The surface is smooth, opaque, and has a pale ocher color. No diffusible dye is produced.

■Juice agar slant culture: Shows medium filamentous growth, is opaque, and has a pale ocher color.

■ Meat juice liquid culture, ij, , raw green in a mixed well, small nail sink V
No bacteria are formed at all.

■ Meat t Tosefchin Hibikiji...j Cultivation 3. Do not liquefy Zeladun.

■ Ritmanu Miruri: Bepu I Nyih not.

(c) Physiological properties ■ Nitrate loss; bt'8j meter (denitrification reaction: Kazushi's note ■ MR (methyl red) test, [feedability ■ vP (Vouge 7 experiment) ■ Formation of indole: Intoxication ('0 Generation of hydrogen sulfide - starch crab) Moisture j'f:' : 'j';,'
j j Raw (, Utilization of citric acid: Positive (f! 41 Utilization of mud nitrogen sources I) Potassium nitrate; Positive ■) Ammonium sulfate: υ-1 [phase] Formation of pigment: Pale pink diffusible pigment generate.

q Liurease: Positive 0 Oxidase: Positive [Phase] Catalase: Positive (weak) ■Growth range T) pH: Grows at pH 4.0 to 7.0, but optimum pH
H is 4.6-5.5° 11) 2 degree of clogging: grows at 14-34°C, but the optimum temperature is 2
8-34℃.

(Attitude to be carved into 0 acid tension: aerobic (1Ω0-F (oxidative bufermentative)
Test (Hugh Leifs)
on) method]: Production of acids and gases from oxidative crushing: Acid Gas L-arabinose − −D-xylose − − D-glucose − D-mannose ± − D-fructose − − D-galactose + −α Gas malt sai j + j - one shot ゛wa, 4
10 - Breasts
] Tang ---Trehalose
--D-sol bit 1・
--D-Mannit --- Inosit
−−Glycerin
--Den Bun
- The uK-+521 strain, which has the following mycological properties, was obtained using the Bersey's Manual
Of Decuminative Bacteriology (Berg)
ey's Manua EngineeringOfDeterminati
ve Bacteriology) Since there is no annotation in the 8th edition, it was identified as Spirillum jmanwa 1 bacterium.
-1521m by Nupirilum NP UK-1521
The strain was named Spirillum sp, UK-1521.

In addition, this strain UK-1521 has been used since May 6, 1981 by the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry (FB).
I) K accession number FEB, MP-7075, and IFO to Fermentation Research Institute from February 9, 1988.
14227, respectively.

The properties of the Spirillum bacteria used in the present invention are generally susceptible to changes, such as exposure to ultraviolet rays and X-rays.

It can be easily mutated by artificial alienation methods using chemical products (e.g., nitrosoguanidine, ethyl methanesulfonic acid), etc., and any mutant strain of TAN-458, which is the subject of the present invention, is Anything with full production capacity can be used in the present invention.

When culturing TA N-458 producing bacteria, examples of carbon sources include glucose, sucrose, maltose, corpse molasses, glycerol μ, oils and fats (e.g. soybean oil, olive oil, etc.), organic acids (e.g. citric acid, succinic acid,
Gluconic acid, etc.) that can be assimilated by bacteria are used as appropriate. As a nitrogen source, for example, soybean powder,
Corn Nuteep Liquor, Dry) Mulberry yeast, yeast extract, meat extract, peptone, urine memo, ril, ammonium acid, ammonium nitrate, ammonium chloride, ammonium phosphate, etc. Compounds and inorganic sulfur compounds are used. can. In addition, Hunchu salt includes inorganic salts normally cultivated by bacteria, such as sodium chloride, potassium chloride, μsium carbonate, magnesium sulfate, potassium phosphate, and disodium phosphate, either singly or in appropriate combinations. used.

In addition, antibiotics T A N
Assimilated sulfur compound proboscis of -458 production orchids, such as (kate salts (e.g., ammonium sulfate, etc.), thiodentagen salts (e.g., thiosulfate [ammonium, etc.),
Inorganic sulfur compounds such as ammonium sulfite) + sulfur-containing amino acids (e.g. synutin, cysteine).

L-thiazolidine-4-carvone 1. When organic sulfur compounds such as IQ)+hypotaurine, 1f peptides (eg, glutathione), or mixtures thereof are added to the medium, the amount of the target product produced may be increased.

In addition, metals such as ferrous sulfate and copper sulfate, and vitamin B
Vitamins such as l, biotin, etc. are also added as necessary. Furthermore, antifoaming agents and surfactants such as silicone oil and polyalkylene glycol ether μ may be added to the medium. Helps the growth of bacteria, TAN-45
Rooted substances or inorganic substances that promote the production of No. 8 may be added as appropriate.

The culturing method may be the same as a general antibiotic production method, and solid culture or hedron culture may be used. For liquid culture, static culture and agitation culture are used.

Although any method such as shaking and culturing may be carried out, aerated agitation culture is particularly preferred. The culture temperature is preferably in the range of about 10 to 35 tE, the pH of the medium is in the range of about 4 to 8, and the culture is carried out for about 8 to 168 hours, preferably 24 to 144 hours.

To collect the target antibiotic 1TAN-458 from the culture, any means normally used to collect metabolites produced by microorganisms from the culture of the microorganisms may be used as appropriate. For example, TAN-458 is a water-soluble acidic substance and is mainly contained in the P solution, so after removing the bacterial cells by filtration or centrifugation, the effective substance is generally separated and purified from the aqueous solution.

Use the method of collecting. That is, A6-exchange chromatography, molecular sieve chromatography, vacuum chromatography, etc. may be used alone or in combination with an appropriate solvent.
Used in combination or repeatedly. Adsorption c: 1 is activated carbon, absorbent resin, anion exchanger 1d
Anio type), powdered cellulose, silica gel, etc. are used, or a single-organic carrier such as a molecular sieve is also used.

The bathing solvent varies depending on the carrier [・≦−f1, but for example, water-soluble solvent, acetone, methanol, ethanol, propatool, butanol, isopropanol,
Contains such as isobuknol or alkaline!
J + 7,55 荊敲 or 葡 t; or aqueous organic salts are used.

More specifically, after completion of the culture, the culture solution is centrifuged to remove the light body. Since the substance is an acidic substance, anion exchange resin [Amberlite, Engineering RA-400
, 4,02,410 (manufactured by Rohm and Haas, USA), DOWEX-1 (manufactured by Dow & Chemical, USA), Diaion 5A-21A and C (manufactured by Mitsubishi Kasei), etc. The 01- or ACO-type can be advantageously used.

The eluate of the adsorbed substance is saline or buffer (41
The active substance is eluted using Chromatography using activated carbon was performed to desalt the eluate, and water-containing alcohol/l/
Elutes with etc. Further, even better results can be obtained by adding dilute ammonia water or the like to elute in neutral or slightly basic conditions.

The eluate of the active substance was concentrated under reduced pressure, and the concentrated solution was treated with an anion exchange molecular sieve resin, QAE-Sephadex A-2.
5 (Cl type, manufactured by Pharmasha, Sweden), and eluted with saline and fractionated into one fraction. When the active fraction is subjected to activated carbon chromatography and desalted, the compound ■
A solution containing 11 rum is obtained. Furthermore, instead of using Sephadex, the antibiotic in the concentrate can be extracted by a method commonly called ion-paired extraction. That is, the active ingredients of the n active ingredients are extracted with a solution of halogenated hydrocarbon containing quaternary Apki/L/(or Bendi/1/) ammonium halide. For example, tri-n-octyl methyl ammonium chloride, n-hexadecylbenzyldimethyl ammonium chloride, tetra-
Examples include n-pentylammonium bromide. These ammonium salts are generally used in a concentration of 0.5 to 6%, particularly preferably 1 to 2%. Regarding halogenated hydrocarbons, for example, methylene
Dichloride, chlorophorem, etc. are used as solvents. The extract contains alkali metal halides (e.g., sodium iodide, potassium iodide, sodium bromide, potassium bromide) or alkaline earth metal halides (e.g., magnesium iodide, zinc iodide, calcium iodide, magnesium bromide, When treated with an aqueous solution containing calcium bromide), the antibiotic is recovered as an alkali metal salt or an alkaline earth metal salt in the aqueous solution.

The alkali metal halide or alkaline earth metal halide foot is between 1.1 and 1.1 of the quaternary ammonium halide used.
2.0 molar equivalent may be used.

The resulting aqueous solution is subjected to chromatography using activated carbon or an adsorbent resin such as Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation) and desalted to obtain a solution containing the alkali metal salt or alkaline earth metal salt of compound (1). . If the antibiotic still contains impurities using the methods described above, the methods described above are used. That is, an aqueous solution containing Compound I is subjected to FAW (j), and the concentrate is subjected to preparative high-performance liquid chromatography for further purification. μ [manufactured by Toyo Soda Co., Ltd.], YMC gel [
[manufactured by Kyoto Chromato Co., Ltd.] etc. are used.

As the mobile phase, an aqueous solution containing reagents used in the ion-paired method, such as tetra-n-butylammonium hydroxide, detofu n-gutiammonium phosphate, etc., is used. The fractions are analyzed for uniformity by analytical high performance liquid chromatography, and both fractions showing a single peak are collected.

When all sodium iodide or sodium bromide is added to this fraction, Compound I becomes the sodium salt. The resulting solution? After subjecting to activated carbon chromatography and desalting, the eluate is made into a paste at low temperature under completely evaporated pressure, and diethyl ether or the like is added to the concentrate to obtain the powdered sodium salt of compound (1). However, the eluate should be concentrated and the glue solution should be
When dried, the sodium salt of the compound is immediately treated.

TAN-458's (-C ton style is T A 1q -458
This is easily confirmed by comparing the nuclear magnetic resonance spectra ()i-NMB) of sodium 2a and 5Q2786o p-nitrobenzyl ester (SQ27860pIqn and ν; each) (Table 1).

(Left below) Table 1 δppm J (Hz) * W, L, Parker et al, J, A
ntibiotics 35+653 (1982
) Also, TAN-458 has one asymmetric element, but the absolute coordination is expressed by the following formula, which is the known carbapenem antibiotic C-19393u2M2! It can be easily inferred by comparing the circular dichroism (CD) spectrum.

In the CD spectrum of TI state-458C-19393H2142, that is, the sodium salt of compound engineering, [θ]
12°218 (-12,100), 1n and 255 (+7,510) all exhibit the characteristic Kotton effect, while that of C-+9393H2M2 [
θ〕'12°230 (-27300) and 253n
Mouth (+23100) LS, Harada et al.+Tetra
hedron +39, γ5 (+983)) shows a trickling effect.

These have the same CD spectrum pattern as eton,
(The absolute configuration of the 5th position of TAN-458 is I
? It is estimated that it is 5.

The physicochemical properties of TA xq-458 sodium salt obtained in Example 2 described below are as follows.

(1) Shape: White powdery substance (2) Molecular formula (molecular weight) i C7H6N03Na (1
75,12) (3) Ultraviolet absorption spectrum) Enter v (in water) 253 ± 2 nm (Fi1% = 156
NmaX l! (4)
Infrared absorption spectrum yv<on salt plate), main peak
(l:17!-1) Two 3450. 2960.
1750°+580.1410.1340.1290
．． 1260°1190.1130.1050,1020
,950°895, 840. 800. 780.
750°710, 660 (5) 1H-117MI'l Spect N (400'
Hz, D20) (δppmJ(H,) 6.2
9 (IH, t, J=2.6.H2).

4.30 (IH5m, H5), 3.46 (IH, dd
, J-5,4゜16.8. H6a), 3.04(I
H, dd, J=2-9.16.8°H6b), 2-9
0 (IH, dda, J = 18.8.2.6, 9.6°Ja), 2.77 (IL add, J = 18.8.2
．． 6.8.4°Hlb) (6) CD spectrum (in water); [θ] nm 218 (-12,100) and [θ)
255 (+ 7,510) m (7) i6 M chromatography [cellulose f
2f41.

(manufactured by Raiko Kasei Co., Ltd.)] Solvent system Efn-propertool: water (4:1) 0.46 (
8) High performance liquid chromatography (manufactured by Waterne,
USA) 1. l! , ii body, Micro Bonder Pack C,,
, mobile phase, 0.0057A tetra-n-1 tylammonium high I゛loxide 10.02) Δphosphorus t'j?
M concentration (pH 7,8), 2 qi/min
Rt (min) = 8.5 (9) Solubility: Easily soluble in water, dimethyl nufoxide, etc.

00 Stability: Decomposes immediately in a water bath with a pH of 5 or higher and in methanol in a solution (in a package)
H7, 5-9) It is stable in the middle.

Next, the biological properties of TAN-458 will be described. T
What is the anti-inflammatory spectrum of A N-458 STLM salt against various feeding organisms? : It is recommended to show it in the Ffj2 table. As is clear from this table, TAN-458 is effective against Durham-positive and Durham-negative cases.

■ 0 Φ self to ■ 0 he to ■ of O■ of -
Sa1 ω('l') COCo C)
0 to N ■ O■ to ω
As is clear from the above data, TAN-458 obtained by the present invention exhibits a strong growth-inhibiting effect on rutting organisms such as Durum-positive bacteria and Durum-negative bacteria. Therefore, T
AN-458 can be used as a fungicide in the treatment of NI bacterial infections in humans and livestock (eg, cows, geese, chickens), etc.

To use TAN-458 as a therapeutic agent for E. coli infection, for example, TAN-458 sodium salt can be dissolved in total physiological saline and administered parenterally subcutaneously or intramuscularly as an injection at 1 to 1 OOlng/kq. /day, preferably 5 to 501197 days for 7 days.

In addition, as an oral preparation, rAy-458 is mixed with lactose to form a capsule, and TAN-458 is prepared from 1 to 1001'!
g/kq/day, preferably 5-50 kg/kg/day.

Furthermore, TAN-458 is an extremely promising compound as a synthetic intermediate for new medicines or as a dream come true.

Next, the present invention will be explained in more detail with reference to specific examples, but the present invention is not limited thereto.

Basen 1- indicates claw J capital/volume Iit% without particular mention.

Example 1 Spirillum sp. U grown on nutrient agar slant mi
K-1521 (, FERM P-7Q75,
FO14227) strain, glucose 2%, soluble ζ
5 star flour 3%, soybean flour 1%, corn nutip liquor 1%, polypeptone (manufactured by Otama Nutritional Chemical Co., Ltd.) 0.5%, dietary tom 0.3%, precipitated TFI calcium carbonate 0.3% ( Seven Erlen-Meyer flasks were inoculated with 200 * containing 40 rnt of a medium consisting of 3% glucose (pH 6.5) and cultured at 24°C for 48 hours. Acupuncture powder 3%, Proflo (manufactured by Brokku & Gamble) 1.5%, Soy Bean Meat TV 1, 5%, cobalt chloride 2p
pm (pH 6,5) containing 2 Otzl
00vl Mulen Meyer flask 250 bottles K,
nj+ g type rHol ct Tutu connection 11 L, 17℃
We carried out 4-f/mata-mine cultivation for 3 days. The culture was centrifuged at low temperature to yield a supernatant of approximately 7.5e.

Add all acetone (<30 g) to the supernatant synovial fluid, stir, and filtrate. Concentrate the obtained acetone extract and transfer the concentrated branch (71) to column chromatography using DOWEX IX2 (ci-type) (200 rsl). Attached. Valid category is 012
Elute with J saline, correct to pl, 5, and prepare on a column of activated carbon (400 d) that has been pretreated with dilute ammonia water. The mixture was eluted and fractionated with water and 20% Meknol water. The eluate was concentrated +li'4 L, 1 night (7
50gl) was extracted with 2% tri-n-octylmethylammonium chloride/methylene chloride solution and re-extracted with methylene chloride extracted MTh1.5% sodium iodide aqueous solution (50Or+J).

The pH of the extract was adjusted to 8, and the adjusted solution was subjected to column chromatography using activated carbon (150 ml) prepared in the same manner as above. Both fractions eluted with water were subjected to analytical HPLC, and the fraction showing a single peak was distilled under reduced pressure at low temperature to obtain an oily sodium salt (3C1').

Example 2 A swollen cultured green surface was cultivated with large / l and the same vermilion as in Example 1 (
Cardiac supernatant fluid (3.5 g) was treated with i2L'' in the same manner as in Example 1, and the eluate of the first active chroma 1-graph total QAE-Sef1Dex A-25 (CI Type.

The mixture was subjected to column chromatography using Farmana Co., Ltd. (Tai, Sweden) (50 yt). Antibiotics Q, l
Elution was continued for 5 minutes with M saline. Effective IZ minute (45yt)
was subjected to active m chromatography (50 el, and 9 fractions were eluted with water.
For preparative high-performance liquid single-bottle chromatography using JY-ri as TSK-gel (Chuunrenshugomekogohan') as Zeyu-body.
Attached, 0.005M tetragutylammonium hydroxide / 0.02 yh phosphorus le'Lj i2J'
It was eluted with ttIIJ solution (pH 7, 8). Valid category (5
5) Sodium iodide (41'V) was added to the instep, and the aqueous solution was subjected to activated carbon chromatography. 1) Distill the mixture under reduced pressure at low temperature, and then add diethyl ether to the 7j:J 4'l'f product and it will combine! ! Is the sodium salt of 2Fe (22...y) a hygroscopic tree? I was bitten.

[Brief explanation of the drawing]

Pass + im is the infrared absorption spectrum (on a salt plate) of T/view N-458 sodium salt, and Figure 2 is H-) I
↑1R spectra (400MH2, volumetric water) are shown.

Claims (1)

[Scope of Claims] A compound of the above formula, which is characterized by culturing in a whole culture medium a microorganism producing a compound of the formula belonging to the genus Spirillum, producing and accumulating the compound of the above formula in the culture, and collecting the compound. or the method of manufacturing the salt.