DE1065134B - Manufacture and extraction of the antibiotic pyrromycin - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

kl. 30h 6

INTERNAT. KL. C 12 d

PATENT OFFICE

EDITORIAL 1065134

F 26527 IVa / 30h

ANM E L D E T A G:

NOTICE DEK REGISTRATION AND ISSUE OF THE EDITORIAL:

SEPTEMBER 1, 1958

SEPTEMBER 10, 1959

The invention relates to the use of the Streptomycete strain DOA 1205 for the production and extraction of a new antibiotic called pyrromycin.

The pyrromycin-producing strain was isolated from an East African soil sample and is called Streptomyces strain DOA1205 deposited in the Centraalbureau voor Schimmelcultures, Baarn (Holland) and characterized by the following properties:

Microscopic morphology: monopodial trees with spiraled ends, spirals with three to five Turns.

Manufacture and extraction of the antibiotic pyrromycin

Applicant: Farbenfabriken Bayer Aktiengesellschaft, Leverkusen-Bayerwerk

Dr. Hans Brockmann and Dipl.-Chem. Werner Lenk, Göttingen,
have been named as inventors

Cultural characteristics

fertile soil growth Aerial mycelium Soluble pigment Physiological
Reactions Synthetic agar
after Lindenbein strong, crusty, brick
red to wine red slightly floury,
white-gray to
reddish gray brick red to wine red Modified Czapek
Thom broth
Baldacci moderate, reddish reddish gray reddish Glycerine Glycocolla
Agar according to v. Plotho pustular, strong, brick
red to wine red floury, gray to
reddish gray brick red to wine red Glucose asparagine agar
after Lindenbein strong, pustular,
brick red floury, gray to
reddish gray brick red to wine red Calcium Malate Agar
according to Baldacci strong, pustular,
pale reddish brownish gray is missing Strength plate after
Linden bones crusty to pustular,
pale brick red reddish gray is missing Starch hydrolysis:
mediocre Cellulose broth
Linden bones single submerged
colonies, pale reddish is missing is missing Glucose agar
Linden bones weak, crusty,
brownish only on individuals
Make, know up
reddish gray pale greenish Nutrient agar after
Linden bones punctiform to
pimple is missing is missing Potato agar
Baldacci pimply to wrinkled,
wine red floury, reddish gray wine red Oatmeal agar
after sexton punctiform, reddish at first
lich, later bluish mouse gray first pale red, later
pale blue Litmus milk after
Linden bones strong ceiling, first
pale red, later pale
blue is only developing
late, floury,
reddish gray full peptonic
after
21 days

909 627/386

Cultural characteristics

fertile soil growth Aerial mycelium Soluble pigment Physiological
Reactions Serum medium after
Spoonbill crusty, brick-red to
bright red is missing is missing no
liquefaction Carrot slices crusty to wrinkled,
wine red floury, reddish gray Potato slices crusty, later wrinkled,
wine red floury, gray to
reddish gray Gelatin stab culture
after Lindenbein crusty, brick-red is missing is missing liquefaction
slow

Other Streptomycete strains, e.g. Frankl097, Garda 1026, Erf 1432 and Esch 1479, can also be used to produce of pyrromycin can be used. These are similar to the strain DOA 1205, but different z. B. Frank 1097 strain of this on most nutrient media due to the lack of typical aerial mycelium.

The fermentation takes place according to the usual working methods, i.e. in the submerged or surface process using the usual culture media.

The antibiotic can be obtained from the culture filtrate and from the mycelium by extraction and / or adsorption processes. In particular, butanol are suitable for extracting the active substance from the culture solution and acetone to which 0.5 ° / ₀ hydrochloric acid may be added for obtaining the Pyrromycin from the mycelium. After removal of the extraction agent and exhaustive extraction of the resulting brown, greasy residues with petroleum ether and ether to separate ineffective accompanying substances, the pyrromycin can be obtained as a hydrochloride from acetone in fiery red crystals.

Pyrromycin hydrochloride, which according to the elemental analysis has the empirical _{formula C 30} H _{36 ClNO 11} , melts at 162 to 165 ° C (corrected, Kofler block), is easily soluble in methanol, water and pyridine, moderately soluble in chloroform and sparingly soluble in benzene. The solutions in these solvents are brown-red and have an intense green fluorescence. Pyrromycin dissolves violet-red in aqueous alkali, in concentrated sulfuric acid initially with a violet color, which, however, immediately turns blue. In high-voltage electrophoresis, pyrromycin migrates to the anode as a cation in an acidic environment. If the rate of migration of the pyrromycin cation is measured in volts / cm and plotted as a function of p _H , a curve is obtained from which the isoelectric point can be determined _{as p H = 8.7 by extrapolation.}

The antibiotic shows characteristic absorption bands in the visible and ultra-red spectral range. The absorption spectra are shown in Figs. I and 2. Fig. 1 shows the absorption spectrum of pyrromycin in chloroform, Fig. 2 shows the ultra-red spectrum of pyrromycin in potassium bromide (0.630 mg / 200 mg KBr).

Pyrromycin inhibits the growth of Staph, aureus up to a dilution of 1: 500,000.

The action of acetic anhydride in pyridine with the addition of a small amount of p-toluenesulfonic acid chloride turns pyrromycin into a tetraacetate hydrochloride with the empirical _{formula C 38} H _{44 ClNO 15} , [a] | ^J + 52 ° (chloroform), which is easily soluble in methanol, water and chloroform and sparingly soluble in benzene and cyclohexane. Pyrromycin hydrochloride likely has the following formula:

O OH

O OH O OH

CH

CH ₂

CH-NH (CH ₃ ) ₂

CH-OH

i I
CH O

CH ₃

he

Pyrromycin is not identical to any of the known antibiotics. Cinerubin A and B, which are close to pyrromycin, can be differentiated from pyrromycin by spectrophotometry and paper chromatography. Cinerubin A has a specific extinction coefficient (specific extinction coefficient = extinction of a solution of 1 g / 1 in d - 1 cm) of 57.9 in methanol at λ = 234 mn, pyrromycin hydrochloride has a specific extinction coefficient of at the same wavelength in the same solvent 76.6. In a solvent mixture of formamide and benzene, cinerubine B hydrochloride migrates with the solvent front on the paper chromatogram, while pyrromycin hydrochloride moves only a little away from the starting point.

example

In a 1000-1 submerged fermenter, 7001 nutrient solution of the composition: 2.0 _{% / 0} glycerine, 0.25% / ₀ glycocolla, 0.10 ° / ₀ sodium chloride, 0.10 ° / ₀ sec. Potassium phosphate, 0.01 ° / ₀ iron (II) sulfate · 7H ₂ 0,0,01 ° / ₀ Magnesium sulfate · 7H ₂ O, 0.01% calcium carbonate and 2.0 ° / ₀ agar successive two days Sterilized for 20 minutes at 120 ° C. and inoculated with a growing culture of Streptomyces strain DOA 1205.

After a culture period of 4 to 5 days at 30 ° C, while the increases, the p _H of 6.0 to 7.7, the

Claims (2)

Fennentation interrupted, the mycelium spun off, the culture solution in a vacuum circulation evaporator concentrated to 5001 and extracted with 2001 butanol. The red-brown butanol extract is used in vacuo Distilled dry, the residue extracted with 11 chloroform, the chloroform extract shaken with 11 water (Wash water discarded) and the chloroform was removed at a bath temperature of 70 to 75 ° C. It remains behind a brown, greasy mass. The separated, still moist mycelium is treated several times with proportions of 41 acetone, the last 0.5% hydrochloric acid were added, stirred, the combined acetone extracts are neutralized with ammonia, and that Acetone is evaporated. A brown, greasy residue remains, which is the same as that from the culture solution received portion is combined and further processed together. The combined residues are exhaustively extracted with petroleum ether and then with ether Petroleum ether and ether extracts discarded, and the remaining crude pyrromycin (120 g) is with 2.5 1 Extracted chloroform. The evaporation residue of the chloroform extract is digested with water, which Evaporated water extract in vacuo at 45 ° C, the residue taken up in methanol, the same Volume of acetone is added, then precipitated impurities are filtered off and the solvents are evaporated. The remaining greasy, dark brown-red residue is boiled with acetone. At the fast When the acetone extract cools, the pyrromycin hydrochloride falls finely crystalline in fiery red, when slow Cool in dark red crystals. Pyrromycin hydrochloride melts to form a red melt at 162 to 165 ° C (corrected, Kofler block), is easily soluble in water, methanol and pyridine, moderately in chloroform, Slightly soluble in benzene, dissolves violet-red in aqueous alkali, initially in concentrated sulfuric acid violet, then blue, and gives a molecular weight on conductometric titration with perchloric acid in glacial acetic acid of 630. One at 80 ° C for 8 hours at high vacuum-dried pyrromycin hydrochloride gives the following elemental analysis:
C ₃₀ H ₃₆ ClNO ₁₁ (621.5)
Calculated C 57.90, H 5.79, Cl 5.70, N 2.25, 1 CH ₃ O 4.99, 2 CH ₃ - C 4.8, 2 N-CH ₃ 4.8; Found C 56.68, H 6.42, Cl 7.2, N 2.54, CH ₃ O 4.36, CH ₃ -C5.0,
NC H ₃ 2.77. 300 mg pyrromycin hydrochloride are mixed with 10 ecm acetic anhydride, 1 ecm pyridine and with a small amount p-Toluenesulfonic acid chloride stored for 3 hours at room temperature. After adding water this becomes Acetyüerungsprodukt extracted with chloroform, the chloroform evaporated, the residue dissolved in acetone and adsorbed on a silica gel column pretreated with hydrochloric acid. Develop when washing with acetone there are several zones, of which the third from the bottom is mixed with cyclohexane after acetone elution. After short Time the pyrromycin tetraacetate hydrochloride falls in light yellow crystals, [ajf + 52 ° (chloroform). Pyrromycin tetraacetate hydrochloride shows no sharp melting point, is easily soluble in methanol, water, chloroform and sparingly soluble in benzene and cyclohexane. A preparation dried at 80 ° C for 8 hours in a high vacuum gives the following elemental analysis: C ₃₈ H ₁₄ ClNO ₁₅ (789.5) Calculated C 57.80, H 5.57, N 1.77, Cl 4.5, 4 CH ₃ CO 21.8; found C 58.17, H 6.00, N 1.70, Cl 3.4, CH ₃ CO 22.4. PATENT CLAIM: Use of the Streptomyces strain DOA 1205 for the production of the antibiotic pyrromycin the usual biological route and extraction of the antibiotic from the fermentation liquids or the mycelium by adsorption and / or extraction 1 sheet of drawings © ι 909 627/386 9. 59 '