DK141999B - Process for the preparation of antibiotic substance designated 30,504 RP, its metal salts and its salts with nitrogenous bases. - Google Patents
Process for the preparation of antibiotic substance designated 30,504 RP, its metal salts and its salts with nitrogenous bases. Download PDFInfo
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(11) FREMLÆG6 ELSESSKRIFT 141999 (¾) DANMARK (51) lnt 0, 3 c 12 p ^,06 L-TMIM ΐνίΛΛΓΤίν C gy g jj/qq «(21) Ansøgning nr, 5Ο65/77 (22) Indleveret den 15·· nov · 1977 (23) Løbedag 28. feb. 1975 (44) Ansøgningen fremlagt og fremlæggelsesskriftet offentliggjort den 4» SUg· 1980 DIREKTORATET FOR ^ PATENT-OG VAREMÆRKEVÆSENET ^ Prioritet begæret fra den(11) SUBMISSION 6 ELSCRIPTION 141999 (¾) DENMARK (51) lnt 0, 3 c 12 p ^, 06 L-TMIM ΐνίΛΛΓΤίν C gy g jj / qq '(21) Application no. 5Ο65 / 77 (22) Filed on 15 · · Nov · 1977 (23) Race day 28 Feb 1975 (44) The application presented and the petition published on 4 'Suk · 1980 DIRECTORATE OF THE PATENT AND TRADEMARKET ^ Priority requested from the
1. mar. 1974, 7407066, FRMar 1 1974, 7407066, FR
(71) RHONE-POULENC INDUSTRIES, 22, Avenue Montaigne, 75560 Paris Cedex 08, FR.(71) RHONE-POULENC INDUSTRIES, 22, Avenue Montaigne, 75560 Paris Cedex 08, FR.
(72) Opfinder: Jean Florent, 6, rue Raffaelli, Paris 16erne, FR: Jean Runel, 6, rue de Toul,"Paris 12eme, FR: Denise Mancy, 7 bis avenue Stinvil= le, Charenton (Val-de-Marne), FR.(72) Inventor: Jean Florent, 6, rue Raffaelli, Paris 16s, FR: Jean Runel, 6, rue de Toul, "Paris 12eme, FR: Denise Mancy, 7th Avenue Stinvil = le, Charenton (Val-de-Marne) ), FR.
(74) Fuldmægtig under sagens behandling:(74) Plenipotentiary in the proceedings:
Ingeniørfirmaet Budde, Schou&Co.__ (64) Fremgangsmåde til fremstilling af antibiotisk stof betegnet 50.5°4 RP, dets metalsalte og dets salte med nitrogenholdige baser.The engineering firm Budde, Schou & Co. (64) Process for the preparation of antibiotic substance designated 50.5 ° 4 RP, its metal salts and its salts with nitrogenous bases.
Den foreliggende opfindelse angår en fremgangsmåde til fremstilling af et hidtil ukendt, antibiotisk stof, der i det følgende betegnes ved nummeret 30.504 RP, dets metalsalte eller dets salte med nitrogenholdige baser.The present invention relates to a process for the preparation of a novel antibiotic substance, hereinafter referred to as the number 30,504 RP, its metal salts or its salts with nitrogenous bases.
30.504 RP er af ganske særlig interesse som følge af dets virkning mod coccidier, der manifesterer sig ved siden af dets antibakterielle virkning og dets virkning mod malaria.30,504 RP is of particular interest as a result of its action against coccidia manifesting alongside its antibacterial and anti-malarial effects.
Fremgangsmåden ifølge opfindelsen er ejendommelig ved, at en stamme af Streptomyces gallinarius DS 25.881 (NRRL 5.785) dyrkes aerobt på et sædvanligt egnet medium og under betingelser, der er sædvanlige for denne kulturtype, hvorefter 30.504 RP, der er dannet under dyrkningen, fraskilles, eventuelt i form af et salt, og renses.The method according to the invention is characterized in that a strain of Streptomyces gallinarius DS 25.881 (NRRL 5.785) is grown aerobically on a usually suitable medium and under conditions usual for this type of culture, after which 30.504 RP formed during cultivation is separated, optionally in the form of a salt, and purified.
141999 2 30.504 RP er karakteristisk ved, at det har følgende fysiske og kemiske egenskaber: udseende: hvidt, mikrokrystallinsk pulver, opløselighed; 30.504 RP er letopløseligt i chlorerede opløsningsmidler, såsom methylenchlorid og chloroform, i alkoholer såsom methanol og i dimethylformamid, og det er relativt opløseligt i acetone og etliylacetat, tungtopløseligt i hexan og praktisk talt uopløseligt i vand (betegnelserne er ifølge Pharraacopée Francaise IX udgave, 2. del, side 13, (1972), procentjsk sammensætning: 30.504 RP indeholder carbon, hydrogen og oxygen. Dets procentiske sammensætning er ca.: C 69,4%, H 9,0%, O 21,0%, neutraliseringsækvivalent: 30,504 RP er et stof med sure egenskaber, hvis neutraliseringsækvivalent (bestemt i dimethylformamidopløsning ved titrering med tetrabutylammoniumhydroxid) er 830, smeltepunkt: (bestemt på en Kofler-bænk): nær 200°, drejningsevne: (bestemt på en 1%'s opløsning i methanol) den specifikke drejningsevne af 30.504 RP er ca.: [α]2°=-790 [α]206=-188° [α]^5 = -378° ultraviolet spektrum: 30.504 RP har ingen . karakteristisk absorption i det ultraviolette område, infrarødt spektrum: (bestemt ved hjælp af kaliumbromidtabletter)141999 2 30,504 RP is characterized in that it has the following physical and chemical properties: appearance: white, microcrystalline powder, solubility; 30,504 RP is readily soluble in chlorinated solvents such as methylene chloride and chloroform, in alcohols such as methanol and in dimethylformamide, and it is relatively soluble in acetone and ethyl acetate, heavily soluble in hexane and virtually insoluble in water (the terms are according to Pharraacopée Francaise IX Part, page 13, (1972), percent composition: 30,504 RP contains carbon, hydrogen and oxygen, its percent composition is about: C 69.4%, H 9.0%, O 21.0%, neutralization equivalent: 30.504 RP is a substance with acidic properties whose neutralization equivalent (determined in dimethylformamide solution by titration with tetrabutylammonium hydroxide) is 830, m.p.: (determined on a Kofler bench): near 200 °, turning ability: (determined on a 1% solution in methanol ) the specific rotational power of 30.504 RP is about: [α] 2 ° = -790 [α] 206 = -188 ° [α] 5 = -378 ° ultraviolet spectrum: 30.504 RP has no characteristic absorption in the ultraviolet range. , infrared spectrum: (determined using potassium bromide tablets)
Dette spektrum er vist på tegningen, hvor der udad abscissen dels er angivet bølgelængden i μ (øvre skala) og dels bølgetallet i cm ^ (nedre skala) og udad ordinaten den optiske tæthed.This spectrum is shown in the drawing, where outwardly the abscissa is indicated partly the wavelength in μ (upper scale) and partly the wavelength in cm ^ (lower scale) and outward the ordinate the optical density.
I den følgende tabel I er dette produkts vigtigste absorptionsbånd i det indfrarøde område angivet i bølgetal (cm ^).In the following Table I, the main absorption band of this product in the infrared range is indicated in wavelength (cm 2).
3 1419993 141999
Tabel I.Table I.
3440 P 1325 m 915 m 3030 ép 1315 ép. 880 f 2960 F 1295 ép. 865 tf 2938 F 1272 m 845 tf 2880 F 1225 f 825 f 2855 ép. 1200 tf 792 m 2540 tf 1172 m 775 f 2060 tf 1140 m 735 f 1945 tf 1115 ép. 700 f 1900 tf 1100 ép. 675 ép.3440 P 1325 m 915 m 3030 ép 1315 ép. 880 f 2960 F 1295 ép. 865 tf 2938 F 1272 m 845 tf 2880 F 1225 f 825 f 2855 ép. 1200 tf 792 m 2540 tf 1172 m 775 f 2060 tf 1140 m 735 f 1945 tf 1115 ép. 700 f 1900 tf 1100 ép. 675 ép.
1785 ép. 1085 F 655 f 1705 F 1070 ép. 630 ép.1785 ép. 1085 F 655 f 1705 F 1070 ép. 630 ép.
1632 m 1045 ép. 615 m 1622 ép. 1038 F 565 ép.1632 m 1045 ép. 615 m 1622 ép. 1038 F 565 ep.
1535 tf 1012 m 525 m 1458 F 980 F 495 m 1405 ép. 958 F 465 tf 1380 F 935 m F = kraftig m = middel f = svag tf = meget svag ép. = skulder1535 tf 1012 m 525 m 1458 F 980 F 495 m 1405 ép. 958 F 465 tf 1380 F 935 m F = heavy m = medium f = weak tf = very weak ép. = shoulder
Chromatografisk vandring: 30.504 RP kan karakteriseres ved opstigende tyndtlagschro-matografi på silicagel under anvendelse af to systemer af opløsningsmiddelblandinger : ethylacetat/cyclohexan/vand/butanol (i volumenforholdet 50/50/25/5):Chromatographic migration: 30.504 RP can be characterized by ascending thin layer chromatography on silica gel using two systems of solvent mixtures: ethyl acetate / cyclohexane / water / butanol (50/50/25/5 volume ratio):
Rf = 0,45, methylenchlorid/methanol (i volumenforholdet 94/6): Rf = 0,55. Bacteriostatisk virkning in vitro: 30.504 RP har en bakteriostatisk virkning, der især udøves på visse grampositive bakterier.Rf = 0.45, methylene chloride / methanol (94/6 by volume): Rf = 0.55. Bacteriostatic effect in vitro: 30,504 RP has a bacteriostatic effect that is particularly exerted on certain gram-positive bacteria.
Den bakteriostatiske virkning af 30.504 RP over for et vist antal organismer er bestemt ved en af de fortyndingsmetoder, der sædvanligvis anvendes til dette formål. For hver organisme bestemmes den mindste koncentration af det aktive stof, der under definerede betingelser forhindrer organismens synlige udvikling i et egnet næ 4 141999 ringsmedium. Resultaterne af de forskellige bestemmelser er sammenfattet i den følgende tabel II, hvor de minimale, bakteriostatiske 3 koncentrationer er angivet i mikrogram stof pr. cm af næringsmediet:The bacteriostatic effect of 30,504 RP against a certain number of organisms is determined by one of the dilution methods commonly used for this purpose. For each organism, the smallest concentration of the active substance is determined, which under defined conditions prevents the visible development of the organism in a suitable medium. The results of the various determinations are summarized in the following Table II, where the minimum bacteriostatic 3 concentrations are given in micrograms per gram. cm of the nutrient medium:
Tabel IITable II
Minimale bakteriosta-Minimal bacteriostatic
Undersøgte bakterieorganismer tiske koncentrationer _(i pg/cm3)_Bacterial organism concentrations investigated _ (in pg / cm3) _
Staphylococcus aureus, stamme 209 P - ATCC 6538 P 0,2Staphylococcus aureus, strain 209 P - ATCC 6538 P 0.2
Staphylococcus aureus, stamme Smith 0,5Staphylococcus aureus, strain Smith 0.5
Sarcina lutea - ATCC 9341 0,9Sarcina lutea - ATCC 9341 0.9
Streptococcus faecalis - ATCC 8043 0,1Streptococcus faecalis - ATCC 8043 0.1
Streptococcus pyogenes hemolyticus, stamme Dig (Insitut Pasteur) 0,2Streptococcus pyogenes hemolyticus, strain Dig (Insitut Pasteur) 0.2
Diplococcus pneumoniae, stamme Til (Institut Pasteur) 0,05Diplococcus pneumoniae, strain To (Institut Pasteur) 0.05
Neisseria catarrhalis (A 152, Institut Pasteur) 6Neisseria catarrhalis (A 152, Institute Pasteur) 6
Bacillus subtilis - ATCC 6633 0,2Bacillus subtilis - ATCC 6633 0.2
Bacillus cereus - ATCC 6630 0,1Bacillus cereus - ATCC 6630 0.1
Mycobacterium species - ATCC 607 6Mycobacterium species - ATCC 607 6
Escherichia coli - ATCC 9637 >150Escherichia coli - ATCC 9637> 150
Shigella dysenteriae, Shiga L (Institut Pasteur) >150Shigella dysenteriae, Shiga L (Institute Pasteur)> 150
Salmonella paratyphi A (stamme Lacasse, InstitutSalmonella paratyphi A (strain Lacasse, Institut
Pasteur) >150Pasteur)> 150
Salmonella schottmuelleri (paratyphi B) - (stamme Fougenc, Institut Pasteur) >150Salmonella schottmuelleri (paratyphi B) - (strain Fougenc, Institut Pasteur)> 150
Proteus vulgaris >150Proteus vulgaris> 150
Pseudomonas aeruginosa >150Pseudomonas aeruginosa> 150
Giftighed:toxicity:
For kyllinger er DL^g 85 mg/kg ved en enkelt, peroral indgivelse.For chickens, DL ^g is 85 mg / kg by a single oral administration.
Virkning mod coccidier:Effect against coccidia:
Virkningen mod coccidier er bestemt på kyllinger, der især er blevet inficeret med Eimeria tenella og Eimeria acervulina.The effect against coccidia is determined on chickens that have been particularly infected with Eimeria tenella and Eimeria acervulina.
Virkningen'af 30.504 RP, der er inkorporeret i kyllingernes føde, mod coccidier viser sig ved ugiftige koncentrationer på 0,005-0,04 vægtprocent af produktet i foderblandingen.The effect of RP 30,504 incorporated in the chickens' feed against coccidia is shown by non-toxic concentrations of 0.005-0.04% by weight of the product in the feed mixture.
5 1419995 141999
Virkning mod malaria; 30.504 RP har desuden en virkning mod malaria ved eksperimentelt fremkaldte plasmodiuminfektioner hos mus og kyllinger.Impact against malaria; 30,504 RP also has an effect against malaria in experimentally induced plasmodium infections in mice and chickens.
Organismen, der producerer det antibiotiske stof 30.504 RP, er en stamme af Streptomyces, der er isoleret fra en jordprøve, der er taget i Indien, og denne organisme betegnes ved nummeret DS 25.881. En prøve af denne organisme er deponeret i Northern Regional Research Laboratory of the U.S., Department of Agriculture in Peoria, 111.The organism producing the antibiotic substance 30,504 RP is a strain of Streptomyces isolated from a soil sample taken in India and this organism is designated by the number DS 25881. A sample of this organism is deposited in the Northern Regional Research Laboratory of the U.S., Department of Agriculture in Peoria, 111.
(USA), hvor den er indregistreret under betegnelsen NRRL 5785.(USA), where it is registered under the designation NRRL 5785.
Denne organisme, der har egenskaber, der ikke gør det muligt at identificere den med en allerede beskrevet art, må betragtes som en hidtil ukendt art og har fået betegnelsen Streptomyces gallinarius, DS 25.881.This organism, which has properties that do not allow it to be identified with a species already described, must be considered a novel species and has been named Streptomyces gallinarius, DS 25881.
Denne stamme er blevet isoleret under anvendelse af den sædvanlige metode, der består i at suspendere en lille mængde jord i destilleret, sterilt vand, at fortynde suspensionen til forskellige koncentrationer, og at udsprede et lille volumen af hver fortynding på overfladen i petriskåle, der indeholder en næringsagar. Efter inkubering i nogle døgn ved 26°C, hvorved mikroorganismerne udvikles, fjernes de kolonier, der ønskes isoleret med henblik på en fortsat undersøgelse, og de udsåes på næringsagar for at opnå større kulturer.This strain has been isolated using the usual method of suspending a small amount of soil in distilled, sterile water, diluting the suspension to various concentrations, and dispensing a small volume of each dilution on the surface into petri dishes containing a nutritional agar. After incubation for a few days at 26 ° C, whereby the microorganisms develop, the colonies desired to be isolated for further study are removed and seeded on nutrient agar to obtain larger cultures.
Streptomyces gallinarius, DS 25.881 danner cylindriske til ovale sporer, der måler 1,0-1,2 μ/0,8-1,0 μ. Dens sporoforer er anbragt i klaser, og de sporedannende filamenter, der er bløde og bugtede, oprulles ofte i enderne og antager enten en let ombøjet form eller danner en spiralformig vinding, undertiden flere. På grund af sporedannelsesmåden hører denne stamme til gruppen Retinaculum-Apertum ifølge Pridham's klassifikation.Streptomyces gallinarius, DS 25,881 forms cylindrical to oval spores measuring 1.0-1.2 μ / 0.8-1.0 μ. Its sporophores are arranged in bunches, and the spore-forming filaments, which are soft and curved, are often wound up at the ends, either assuming a slightly bent shape or forming a helical winding, sometimes several. Due to the mode of spore formation, this strain belongs to the Retinaculum-Apertum group according to Pridham's classification.
Streptomyces gallinarius DS 25.881 udvikles godt ved 26°C og ved 37°C, men ikke ved 50°C. Den har et sporedannende luftmycelium med grå farve. Det vegetative myceliums farve går i almindelighed, afhængigt af kulturmediet, fra gul eller gråliggul til brungul eller gulbrun. Bortset fra en særlig produktion af opløseligt, brunligt rosaviolet pigment på en agar ifølge Hickey og Tresner giver organismen på alle kulturmedier, hvor den er blevet iagttaget, en produktion af opløseligt pigment, der i almindelighed har en ringe intensitet, og som går fra brunliggult eller brunliggråt til gulbrunt.Streptomyces gallinarius DS 25,881 develops well at 26 ° C and at 37 ° C, but not at 50 ° C. It has a spore-forming gray mycelium. The color of the vegetative mycelium generally varies, depending on the culture medium, from yellow or greyish-yellow to brown-yellow or yellow-brown. Apart from a special production of soluble brownish rosaviolet pigment on an agar according to Hickey and Tresner, the organism on all culture media where it has been observed yields a production of soluble pigment which is generally of low intensity and which goes from brownish yellow or brownish-gray to yellowish-brown.
I kulturer af organismen, der er inkuberet ved 26°C, har den følgende, biokemiske egenskaber: 6 U1999 produktion, af melanin: negativ produktion af hydrogensulfid: negativ tyrosinase: negativ gelatinesmeltning: positiv udnyttelse af cellulose: positiv dannelse af nitriter ud fra nitrater: kraftigt positiv, både i nitratholdig næringsbouillon og i syntetiske medier hydrolyse af stivelse: positiv kultur på skummetmælk: peptonisering uden koagule ring surhedsgraden stiger ganske let.In cultures of the organism incubated at 26 ° C, it has the following biochemical properties: 6 U1999 production, of melanin: negative hydrogen sulphide production: negative tyrosinase: negative gelatin melting: positive utilization of cellulose: positive nitrite formation from nitrates : strongly positive, both in nitrate-containing broth and in synthetic media starch hydrolysis: positive culture on skimmed milk: peptonization without coagule ring acidity increases quite easily.
Kulturegenskåberne af Streptomyces gallinarius DS 25.881 er sammenfattet i den følgende tabel III. Det er egenskaberne af kulturer, der har nået et godt udviklingsstadie, dvs. 2-3 uger ved 26°C, hvor der ikke er angivet andet. Disse egenskaber er iagttaget på næringsagar og bouillonen der sædvanligvis anvendes til bestemmelse af morphologiske egenskaber af Streptomyces-stammer, idet der ved kulturer på agarmedier anvendes skråagar. Et vist antal af de anvendte kulturmedier er fremstillet ifølge de opskrifter, der er angivet i The Actinomycetes, S.A. Waksman, s. 193-197, Chronica Botanica Company, Waltham, Mass., USA, 1950, og i dette tilfælde er de angivet ved bogstavet W fulgt af det nummer, de har i dette værk. Referencerne eller sammensætningerne af de andre medier er følgende: - Ref. A - "Hickey and Tresner's Agar" - T.G. Pridham et al. - Antibiotics Annual, 1956-1957, s. 950 - Ref. B - "Bennett's Agar" - S.A. Waksman - The Actinomycetes, bind 2, s. 331 - nr. 30 - The Williams and Wilkins Company, Baltimore, 1961 - Ref. C - Opskrift W-23, tilsat 2% agar - Ref. D - "Yeast Extract Agar". T.G. Pridham et al. - Antibiotics Annual, 1956-1957, s. 950 - Ref. E - "Tomato Paste Oatmeal Agar" - T.G. Pridham et al. - Antibiotics Annual, 1956-1957, s. 950 - Ref. F - "Melanin formation medium" - S.A. Waksman - The Actinomycetes, bind 2, s. 333, nr. 42 - The Williams and Wilkins Company, Baltimore, 1961 - Ref. G - W.E. Grundy et al. - Antibiotics and Chem. 2, 401, 1952 - Ref. H - "Inorganic Salts - Starch Agar" - T.G. Pridham et al.-Antibiotics Annual 1956-1957, s. 951 - Ref. I - svarer til opskrift W-l, hvor 3% saccharose er erstattet 7 141999 med 1,5% glucose - Ref. J - svarer til opskrift W-l, hvor 3% saccharose er erstattet med 1,5% glycerol - Ref. K - svarer til opskrift W-18, hvor 3% saccharose er erstattet med 1,5% glucose - Ref. L - svarer til opskrift W-18, hvor saccharose er udeladt og erstattet med små filtrerpapirbånd, der delvis er nedsænket i væsken -Ref. M - "Manual of Methods for Pure Culture Study of Bacteria" -Society of American Bacteriologists - Geneva, N.Y. H^q - 18 - Ref. N - "Plain gelatin" - fremstillet som angivet i "Manual of Methods for Pure Culture Study of Bacteria" - Society of American Bacteriologists - Geneva, N.Y. 11^-18 - Ref. P - Skummetmælkepulver (handelsvare) - rekonstitueret - Ref. Q - Medium, der er angivet til undersøgelse af produktion af H2S af H.D. Tresner og F. Danga - Journal of Bacteriology, 76, 239-244, 1958.The cultural characteristics of Streptomyces gallinarius DS 25.881 are summarized in the following Table III. It is the characteristics of cultures that have reached a good stage of development, ie. 2-3 weeks at 26 ° C, unless otherwise stated. These properties are observed on nutrient agar and the broth usually used to determine the morphological properties of Streptomyces strains, with sloped agar cultures used on agar media. A certain number of the culture media used are prepared according to the recipes set forth in The Actinomycetes, S.A. Waksman, pp. 193-197, Chronica Botanica Company, Waltham, Mass., USA, 1950, and in this case, they are indicated by the letter W followed by the number they have in this work. The references or compositions of the other media are as follows: - Ref. A - "Hickey and Tresner's Agar" - T.G. Pridham et al. - Antibiotics Annual, 1956-1957, pp. 950 - Ref. B - "Bennett's Agar" - S.A. Waksman - The Actinomycetes, Volume 2, p. 331 - No. 30 - The Williams and Wilkins Company, Baltimore, 1961 - Ref. C - Recipe W-23, added 2% agar - Ref. D - "Yeast Extract Agar". T. G. Pridham et al. - Antibiotics Annual, 1956-1957, pp. 950 - Ref. E - "Tomato Paste Oatmeal Agar" - T.G. Pridham et al. - Antibiotics Annual, 1956-1957, pp. 950 - Ref. F - "Melanin formation medium" - S.A. Waksman - The Actinomycetes, Volume 2, p. 333, No. 42 - The Williams and Wilkins Company, Baltimore, 1961 - Ref. G - W.E. Grundy et al. - Antibiotics and Chem. 2, 401, 1952 - Ref. H - "Inorganic Salts - Starch Agar" - T.G. Pridham et al. Antibiotics Annual 1956-1957, p. 951 - Ref. I - corresponds to recipe W-1, where 3% sucrose has been replaced with 1.5% glucose - Ref. J - corresponds to recipe W-1, where 3% sucrose is replaced by 1.5% glycerol - Ref. K - corresponds to recipe W-18, where 3% sucrose is replaced by 1.5% glucose - Ref. L - corresponds to recipe W-18 where sucrose is omitted and replaced with small filter paper tapes partially submerged in the liquid -Ref. M - "Manual of Methods for Pure Culture Study of Bacteria" -Society of American Bacteriologists - Geneva, N.Y. H ^ q - 18 - Ref. N - "Plain gelatin" - prepared as specified in "Manual of Methods for Pure Culture Study of Bacteria" - Society of American Bacteriologists - Geneva, N.Y. 11 ^ -18 - Ref. P - Skimmed milk powder (commodity) - reconstituted - Ref. Q - Medium designated for examination of production of H2S by H.D. Tresner and F. Danga - Journal of Bacteriology, 76, 239-244, 1958.
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Streptomyces gallinarius DS 25.881 har et sæt egenskaber, der ikke falder nøjagtigt sammen med egenskaberne af nogle af de allerede beskrevne stammer, og den må derfor betragtes som en ny art.Streptomyces gallinarius DS 25881 has a set of properties that do not exactly coincide with the properties of some of the strains already described, and it must therefore be considered a new species.
Ved at betragte de arter, der er beskrevet i "The Actinomycetes" (bind 2, S.A. Waksman, The Williams and Wilkins Company, Baltimore, 1961) samt i Bergey's "Manual of Determinative Bacteriology" (7. udgave, The Williams and Wilkins Company, Baltimore, 1957), er den art, med hvilken den logisk kan sammenlignes Streptomyces hygroscopicus. Ligesom denne udvikler S. gallinarius DS 25.881 således et vegetativt mycelium, der i almindelighed er gult eller gråliggult til brungult eller gulbrunt, og den har i almindelighed et gråt, sporedannende luftmycelium. Den danner ikke opløseligt melaninpigment på organiske medier, producerer ikke HjS og smelter langsomt gelatine, på hvilken, den ikke danner opløseligt pigment. På alle sine kulturmedier, såvel syntetiske som organiske, hvor den ikke danner opløseligt pigment, eller hvor den kun danner opløseligt pigment, der almindeligvis har en ringe intensitet, er den brunliggul eller brunliggrå til gulbrun. Ud over disse egenskaber har Streptomyces gallinarius DS 25.881 den egenskab, at den på en særlig måde kan producere et opløseligt, brunligt rosaviolet pigment på et af de medier, hvor den er blevet dyrket (agar ifølge Hickey og Tresner), hvilket af H.D. Tresner og E.J. Backus (Applied Microbiology 4, 243-250, 1956) er blevet erkendt som en egenskab ved talrige stammer, der tilhører arten Streptomyces hygroscopicus og er blevet iagttaget på forskellige medier afhængigt af stammerne. Imidlertid afviger den fra denne art ved to af de tre væsentlige egenskaber, ved hvilke den defineres. Dels svarer formen af dens sporoforer ikke til sporoforerne af arten Streptomyces hygroscopicus, og dels dannes der ikke skinnende, sorte områder i dens sporedannende luftmycelium i noget tilfælde, hvilket er karakteristisk for arten Streptomyces hygroscopicus.By considering the species described in "The Actinomycetes" (Volume 2, S.A. Waksman, The Williams and Wilkins Company, Baltimore, 1961) as well as in Bergey's "Manual of Determinative Bacteriology" (7th edition, The Williams and Wilkins Company , Baltimore, 1957), is the species with which it can logically be compared to Streptomyces hygroscopicus. Like this, S. gallinarius DS 25,881 thus develops a vegetative mycelium which is generally yellow or greyish yellow to brownish-yellow or yellow-brown, and generally has a gray, spore-forming air mycelium. It does not form soluble melanin pigment on organic media, does not produce HjS and slowly melts gelatin on which it does not form soluble pigment. In all its culture media, both synthetic and organic, where it does not form soluble pigment or where it forms only soluble pigment, which is generally of low intensity, it is brownish-yellow or brownish-yellow to yellow-brown. In addition to these properties, Streptomyces gallinarius DS 25,881 has the property that it can in a particular way produce a soluble, brownish rosaviolet pigment on one of the media in which it has been grown (Hickey and Tresner agar), which H.D. Tresner and E.J. Backus (Applied Microbiology 4, 243-250, 1956) has been recognized as a property of numerous strains belonging to the species Streptomyces hygroscopicus and has been observed on various media depending on the strains. However, it differs from this species by two of the three essential characteristics by which it is defined. In part, the shape of its sporophores does not correspond to the sporophores of the species Streptomyces hygroscopicus, and secondly, no shiny black areas are formed in its spore-forming air mycelium in any case, which is characteristic of the species Streptomyces hygroscopicus.
Streptomyces gallinarius DS 25.881 kan ligeledes sammenlignes med Streptomyces aureofaciens, der heller ikke danner melanin, i almindelighed danner et gult til orangegult eller bananligt vegetativt mycelium, og som har et gråt, sporedannende luftmycelium. Desuden danner Streptomyces aureofaciens ikke opløseligt pigment på adskillige kulturmedier, og, når dette er tilfældet, danner den opløselige pigmenter med en gul til brunlig farve, der i almindelighed er temmelig svag. I visse tilfælde farves det vegetative mycelium af Streptomyces aureofaciens rødbrunt til purpur, men i dette tilfælde drejer det sig som et pigment, der ikke difunderer ud 1 mediet, 14 141999 og som på grund af dette ikke kan svare til det opløselige, brunlige, rosaviolette pigment, der kan produceres af Streptomyces gallinarius DS 25.881.Streptomyces gallinarius DS 25,881 can also be compared to Streptomyces aureofaciens, which also does not form melanin, generally produces a yellow to orange-yellow or banana-vegetative mycelium, and which has a gray, spore-forming air mycelium. In addition, Streptomyces aureofaciens does not form soluble pigment on numerous culture media, and, when this is the case, it forms soluble pigments with a yellow to brownish color which is generally rather weak. In some cases, the vegetative mycelium of Streptomyces aureofaciens reddish-brown is stained purple, but in this case it turns out to be a pigment which does not diffuse into the medium, 14 141999 and, as a result, cannot match the soluble, brownish, rosaviolet pigment that can be produced by Streptomyces gallinarius DS 25,881.
Desuden smelter S. aureafaciens ikke gelatine, på hvilken den danner et flødefarvet, vegetativt mycelium, og den hverken koagulerer eller peptoniserer skummetmælk, mens S. gallinarius DS 25.881 derimod smelter gelatine, på hvilken den danner et gulbrunt, vegetativt mycelium, og giver en klar og regulær peptonisering af skummetmælk. Det fremgår altså heraf, at S. gallinarius DS 25,881 ligeledes afviger fra denne sidstnævnte art.In addition, S. aureafaciens does not melt gelatin on which it forms a cream-colored, vegetative mycelium and neither coagulates nor peptonizes skimmed milk, whereas S. gallinarius DS 25,881, on the other hand, melts gelatin on which it forms a yellow-brown vegetative mycelium. and regular peptonization of skim milk. It is thus apparent from this that S. gallinarius DS 25,881 also differs from this latter species.
S. gallinarius DS 25.881's evne til at udnytte forskellige carbonkilder eller nitrogenkilder er blevet bestemt ifølge princippet i metoden ifølge Pridham og Gottlieb (J. of Bact. 56, 1Q7-114, 1948). Udviklingsgraden er blevet iagttaget på det basismedium, der er angivet af forfatterne, idet glucose erstattes med forskellige carbonkilder, og ammoniumsulfat erstattes med forskellige nitrogenkil der. De opnåede resultater er angivet i den følgende tabel: 15 141999The ability of S. gallinarius DS 25.881 to utilize various carbon or nitrogen sources has been determined according to the principle of the method of Pridham and Gottlieb (J. of Bact. 56, 1Q7-114, 1948). The degree of development has been observed on the base medium stated by the authors, with glucose being replaced with different carbon sources and ammonium sulfate being replaced with different nitrogen sources there. The results obtained are given in the following table: 15 141999
Undersøgte udnvttelse Undersøgte Udnyttelse carbonkilder ^_ nitrogenkilder __ D-Ribose negativ NaNO^ positiv D-Xylose positiv NaN02 positiv L-Arabinose positiv (NH4)2S04 positiv L-Rhamnose positiv (NH4)2HPC>4 positiv D-Glucose positiv urinstof positiv D-Galactose positiv L-asparagin positiv D-Fructose positiv glucosamin positiv D-Mannose positiv glycocol positiv L-Sorbose negativ sarcosin negativExploitation Examined Exploitation Carbon Sources ^ _ Nitrogen Sources __ D-Ribose Negative NaNO ^ Positive D-Xylose Positive NaN02 Positive L-Arabinose Positive (NH4) 2S04 Positive L-Rhamnose Positive (NH4) 2HPC> 4 Positive D-Glucose Positive Urea Positive D- Galactose positive L-asparagine positive D-Fructose positive glucosamine positive D-Mannose positive glycocol positive L-Sorbose negative sarcosine negative
Lactose positiv DL-alanin positivLactose positive DL-alanine positive
Maltose positiv DL-Valin positivMaltose positive DL-Valine positive
Saccharose positiv DL-asparaginsyre positivSucrose positive DL-aspartic acid positive
Trehalose positiv L-glutaminsyre positivTrehalose positive L-glutamic acid positive
Cellobiose positiv L-Arginin positivCellobiose positive L-arginine positive
Raffinose negativ L-Lysin positivRaffinose negative L-Lysine positive
Dextrin positiv DL-serin positivDextrin positive DL serine positive
Inulin negativ DL-threonin positivInulin negative DL-threonine positive
Stivelse positiv DL-methionin positivStarch positive DL-methionine positive
Glycogen positiv taurin negativGlycogen positive taurine negative
Glycerol positiv DL-phenylalanin positivGlycerol positive DL-phenylalanine positive
Erythritol negativ L-tyrosin positivErythritol negative L-tyrosine positive
Adonitol negativ DL-prolin positivAdonitol negative DL-proline positive
Dulcitol negativ L-hydroxyprolin positiv D-Mannitol positiv L-tryptophan positiv D-Sorbitol negativ betain negativDulcitol negative L-hydroxyproline positive D-Mannitol positive L-tryptophan positive D-Sorbitol negative betaine negative
Inositol positivInositol positive
Salicin positiv 16 141999Salicin positive 16 141999
Dyrkningen af Streptoxnyces gallinarius DS 25.881 kan gennemføres ved en hvilken som helst aerob dyrkningsmetode på overfladen eiler submerst,. men den sidstnævnte metode foretrækkes af bekvemmelighedsgrunde- Til dette formål anvendes forskellige typer af apparatur, der sædvanligvis anvendes i gæringsindustrien.The cultivation of Streptoxnyces gallinarius DS 25881 can be carried out by any aerobic cultivation method on the surface or most recently. but the latter method is preferred for convenience reasons. For this purpose, various types of apparatus commonly used in the fermentation industry are used.
Der kan især anvendes følgende arbejdsgang til gennemførelse af operationerne:In particular, the following workflow can be used to carry out the operations:
Streptomyces gallinarius DS 25.881 (stamkultur) agarkultur i rvstekolbekultur podekultur til gæring i produktionskultur til gæring.Streptomyces gallinarius DS 25.881 (stem culture) agar culture in rv charcoal culture seed culture for fermentation in production culture for fermentation.
Gæringsmediet Bør i det væsentlige indeholde en assimilerbar carbonkilde og en assimilerbar nitrogenkilde, uorganiske elementer, især chlorider, og; eventuelt vækstfaktorer, idet alle disse stoffer kan tilføres ‘i? form' af veldefinerede produkter eller komplekse blandinger, såsom biologiske produkter af forskellig oprindelse.The fermentation medium should substantially contain an assimilable carbon source and an assimilable nitrogen source, inorganic elements, especially chlorides, and; possibly growth factors, since all these substances can be added 'in? in the form of well-defined products or complex mixtures, such as biological products of different origins.
Som assimilerbare carbonkilder kan der anvendes kulhydrater, såsom glucose, saøcbarose, maltose, dextriner, stivelse eller andre carbon- og hydrpgenholdige stoffer, såsom sukkeralkoholer (glycerol) eller visse organiske syrer, f.eks. mælkesyre eller citronsyre.Carbohydrates such as glucose, saucbarose, maltose, dextrins, starch or other carbon and hydrogen-containing substances such as sugar alcohols (glycerol) or certain organic acids, e.g. lactic or citric acid.
Visse animalske eller vegetabilske olier, såsom spækolie eller sojaolie kan fordelagtigt erstatte disse forskellige carbon- og hydrogenkilder eller anvendes i forbindelse med disse.Certain animal or vegetable oils, such as bacon or soybean oil, may advantageously replace these various carbon and hydrogen sources or be used in conjunction with them.
Der findes overordentlig mange egnede kilder til assimilerbart nitrogen. De.kan være meget simple kemiske stoffer, såsom uorganiske eller organiske ammoniumsalte, urinstof og visse aminosyrer. Nitrogen kan også tilføres med komplekse stoffer, der først og fremmest 17 141999 indeholder nitrogen i proteinform, f.eks. casein, lactalbumin, gluten og disses hydrolysåter, sojamel, jordnøddemel, fiskemel, kødekstrakt, gærekstrakt, distillers' solubles og majsstøbevand.There are an enormous number of suitable sources of assimilable nitrogen. They can be very simple chemical substances, such as inorganic or organic ammonium salts, urea and certain amino acids. Nitrogen can also be supplied with complex substances containing primarily nitrogen in protein form, e.g. casein, lactalbumin, gluten and their hydrolysates, soybean meal, peanut flour, fishmeal, meat extract, yeast extract, distillers' solubles and maize cast water.
Blandt de tilsatte, uorganiske stoffer kan visse have en puffervirkning eller en neutraliserende virkning, såsom alkalimetal- eller jordalkalimetalphosphater eller calcium- eller magnesiumcarbonater. Andre giver den nødvendige, ioniske ligevægt for udviklingen af Streptomyces gallinarius DS 25.881 og for produktionen af 30.504 RP, såsom chlorider og sulfater af alkalimetaller og jordalkalimetaller. Endelig virker visse stoffer mere specielt som aktivatorer for de metaboliske reaktioner i Streptomyces gallinarius DS 25.881, og disse er salte af zink, cobalt, jern, kobber og mangan.Among the added inorganic substances, some may have a buffering or neutralizing effect, such as alkali metal or alkaline earth metal phosphates or calcium or magnesium carbonates. Others provide the necessary ionic equilibrium for the development of Streptomyces gallinarius DS 25,881 and for the production of 30,504 RP, such as chlorides and sulphates of alkali metals and alkaline earth metals. Finally, certain substances act more specifically as activators of the metabolic reactions in Streptomyces gallinarius DS 25,881, and these are salts of zinc, cobalt, iron, copper and manganese.
Vækstfaktorerne er stoffer af vitaminnatur, såsom riboflavin, folinsyre og pantothensyre.The growth factors are substances of vitamin nature such as riboflavin, folic acid and pantothenic acid.
Gæringsmediets pH-værdi ved dyrkningens begyndelse bør være mellem 5,8 og 7,8, fortrinsvis mellem 6,2 og 7,4. Den optimale temperatur ved gæringen er mellem 25 og 30°C, men en tilfredsstillende produktion fås ved temperaturer mellem 23 og 33°C. Beluftningen af gæringsmediet kan varieres inden for temmelig vide grænser. Det har dog vist sig, at beluftninger på 0,3-3 liter luft pr. liter medium og minut er særlig godt egnet. Det maksimale udbytte af 30.504 RP fås efter 2-8 døgns dyrkning, idet dette tidsrum i det væsentlige afhænger af det anvendte medium.The pH value of the fermentant at the beginning of the culture should be between 5.8 and 7.8, preferably between 6.2 and 7.4. The optimum fermentation temperature is between 25 and 30 ° C, but satisfactory production is obtained at temperatures between 23 and 33 ° C. The aeration of the fermentation medium can be varied within fairly wide limits. However, it has been found that aerations of 0.3-3 liters of air per liters of medium and minute are particularly well suited. The maximum yield of RP 30,504 is obtained after 2-8 days of cultivation, this time being essentially dependent on the medium used.
Det fremgår af det foregående, at de generelle betingelser for dyrkning af Streptomyces gallinarius DS 25.881 til produktion af 30.504 RP kan varieres inden for et stort område og tilpasses enhver speciel omstændighed.It is apparent from the foregoing that the general conditions for cultivation of Streptomyces gallinarius DS 25,881 for the production of 30,504 RP can be varied over a wide range and adapted to any particular circumstance.
30.504 RP kan isoleres fra gæringsblandinger på følgende måde:30.504 RP can be isolated from fermentation mixtures as follows:
Blandingen filtreres ved en pH-værdi i det sure område, i almindelighed mellem 3 og 6 og fortrinsvis nær 5, i nærværelse af et filtreringshjælpemiddel.The mixture is filtered at a pH in the acidic range, generally between 3 and 6 and preferably near 5, in the presence of a filtration aid.
Mængden af aktivt stof, der tilbageholdes i filterkagen, ekstraheres ved hjælp af et egnet, organisk opløsningsmiddel, f.eks. en lavere alkohol, såsom methanol, eller et chloreret opløsningsmiddel, såsom methylenchlorid. Råproduktet kan isoleres fra de oven for nævnte organiske opløsninger ved krystallisation efter koncentrering af disse opløsninger under formindsket tryk, idet der eventuelt 18 141999 tilsættes et dårligt opløsningsmiddel eller et ikke-opløsningsmiddel og henstilles koldt. . .The amount of active substance retained in the filter cake is extracted by a suitable organic solvent, e.g. a lower alcohol such as methanol, or a chlorinated solvent such as methylene chloride. The crude product can be isolated from the above-mentioned organic solutions by crystallization after concentration of these solutions under reduced pressure, optionally adding a poor solvent or a non-solvent and leaving it cold. . .
30.504 RP kan renses ved sædvanligt anvendte metoder, såsom omkrystallisation, chromatografering på forskellige,adsorberende midler eller modstrømsfordeling.30,504 RP can be purified by commonly used methods such as recrystallization, chromatography on various adsorbents or countercurrent distribution.
Opfindelsen illustreres ved det følgende eksempel.The invention is illustrated by the following example.
Eksempel.Example.
a) Gæring: I en gæringsbeholder på 170 liter anbringes der:(a) Fermentation: In a fermentation tank of 170 liters:
Pepton: 1200 g Gærekstrakt: 600 gPeptone: 1200 g Yeast extract: 600 g
Glucose-monohydrat: 1200 gGlucose monohydrate: 1200 g
Delvis hydrolyseret stivelse: 2400 gPartially hydrolyzed starch: 2400 g
Ledningsvand op til 110 liter.Tap water up to 110 liters.
3 pH-værdien indstilles til 7,3 ved tilsætning af 50 cm 10 N natriumhydroxidopløsning. Mediet steriliseres ved gennemblæsning med damp med en temperatur på 122°C i 40 minutter. Efter afkøling er mediets volumen 120 liter på grund af kondensationen af damp under sterilisationen, og pH-værdien er 6,90.3 adjust the pH to 7.3 by adding 50 cm of 10 N sodium hydroxide solution. The medium is sterilized by purging with steam at a temperature of 122 ° C for 40 minutes. After cooling, the volume of the medium is 120 liters due to the condensation of steam during sterilization and the pH is 6.90.
Der podes med 200 cm^ af en rystekolbekultur af Streptomyces gallinarius DS 25.881. Kulturen udvikles i løbet af 21 timer under omrøring og beluftning med steril luft. Den er derefter egnet til podning af produktionskulturen.200 cm 2 of a shake flask culture is seeded by Streptomyces gallinarius DS 25,881. The culture is developed over 21 hours with stirring and aeration with sterile air. It is then suitable for grafting the production culture.
Produktionskulturen fremstilles i en gæringsbeholder på 800 liter, i hvilken der er anbragt følgende stoffer:The production culture is prepared in a fermentation tank of 800 liters in which the following substances are placed:
Destillers' solubles: 12,5 kgDistillers' solubles: 12.5 kg
Saccharose: 12,5 kgSucrose: 12.5 kg
Natriumchlorid: 2,5 kgSodium chloride: 2.5 kg
Magnesiumsulfat-heptahydrat: 1,0 kgMagnesium sulfate heptahydrate: 1.0 kg
En opløsning af 20 g cobalt- chlorid-hexanydrat pr. liter 0,5 liter Ledningsvand op til 460 liter.A solution of 20 g of cobalt chloride hexanyrate per 0.5 liters of tap water up to 460 liters.
141999 19141999 19
Mediets pH-værdi indstilles til 7,5 ved tilsætning af 720 cm3 10 N natriumhydroxidopløsning, og det steriliseres derefter ved gennemblæsning med damp med en temperatur på 122°C i 40 minutter.The pH of the medium is adjusted to 7.5 by the addition of 720 cc of 10 N sodium hydroxide solution, and it is then sterilized by purging with steam at a temperature of 122 ° C for 40 minutes.
Efter afkøling er mediets volumen 500 liter på grund af kondensationen af damp under sterilisationen, og mediets pH-værdi er 6,40. Der podes med 50 liter af den ovenfor beskrevne podekultur, der er fremstillet i gæringsbeholderen på 170 liter. Kulturen udvikles i løbet af 116 timer ved 27°C under omrøring med en turbine, der roterer med 205 omdr./min., og beluftning med 20 m steril luft pr. time. Sænkningen af pH-værdien ved dyrkningens begyndelse begrænses til 6,5 ved automatisk tilsætning af 5 N natriumhydroxidopløsning (total anvendt mængde: 0,5 liter).After cooling, the volume of the medium is 500 liters due to the condensation of steam during sterilization and the pH of the medium is 6.40. Inoculate with 50 liters of the above-described seed culture prepared in the 170 liter fermentation vessel. The culture is developed over 116 hours at 27 ° C with agitation with a turbine rotating at 205 rpm and aeration with 20 m sterile air per minute. hour. The lowering of the pH at the beginning of the cultivation is limited to 6.5 by the automatic addition of 5 N sodium hydroxide solution (total quantity used: 0.5 liter).
Efter dyrkningens tilendebringelse er kulturens pH-værdi 7,85, og volumenet er 470 liter.After completion of the culture, the pH of the culture is 7.85 and the volume is 470 liters.
b) Ekstraktion: 1000 liter af gæringsblandingen, der fremstilles under de ovenfor angivne betingelser, omrøres i 1/2 time efter syrning til en pH--værdi på 5 ved tilsætning af 7 liter 6 N saltsyre.b) Extraction: 1000 liters of the fermentation mixture prepared under the above conditions is stirred for 1/2 hour after acidification to a pH of 5 by the addition of 7 liters of 6 N hydrochloric acid.
Efter blanding med 35 kg filtreringshjælpemiddel filtreres gæringsblandingen på en filterpresse. Filterkagen vaskes på filteret med 200 liter vand, hvis pH-værdi er indstillet til 5 ved tilsætning af 6 N saltsyre.After mixing with 35 kg of filter aid, the fermentation mixture is filtered on a filter press. The filter cake is washed on the filter with 200 liters of water, the pH of which is adjusted to 5 by the addition of 6 N hydrochloric acid.
Filtratet og vaskevæsken fjernes.The filtrate and washing liquid are removed.
Filterkagen findeles i 700 liter methanol. Den fremkomne blan- 3 ding indstilles til en pH-værdi på 7 ved tilsætning af 470 cm 6 N natriumhydroxidopløsning og omrøres i 1/2 time.The filter cake is comminuted in 700 liters of methanol. The resulting mixture is adjusted to a pH of 7 by the addition of 470 cm 6 of sodium hydroxide solution and stirred for 1/2 hour.
Blandingen filtreres på en filterpresse, og filterkagen vaskes på filteret med 100 liter methanol.The mixture is filtered on a filter press and the filter cake is washed on the filter with 100 liters of methanol.
Filtratet og vaskevæsken forenes og koncentreres under formindsket tryk (5-10 mm Hg), således at der fås 60 liter vandigt koncentrat.The filtrate and wash liquid are combined and concentrated under reduced pressure (5-10 mm Hg) to give 60 liters of aqueous concentrate.
Dette koncentrat indstilles til en pH-værdi på 3 ved tilsætning 3 af 580 cm 6 N saltsyre og omrøres i 10 minutter med 40 liter methy-lenchlorid. De to faser adskilles. Derefter ekstraheres den vandige fase successivt med 30 liter og 20 liter methylenchlorid.This concentrate is adjusted to a pH of 3 by adding 3 of 580 cm 6 of hydrochloric acid and stirring for 10 minutes with 40 liters of methylene chloride. The two stages are separated. The aqueous phase is then extracted successively with 30 liters and 20 liters of methylene chloride.
Methylenchloridekstrakterne forenes og omrøres med 9 liter 3 vand, idet der syrnes til en pH-værdi på 3 i den vandige fase (20 cm 6 N saltsyre.). Den organiske fase fraskilles og vaskes igen med 18 liter vand, idet der tilsættes natriumhydroxidopløsning til en pH-værdi på 9,5 i den vandige fase (20 cm 6 N natriumhydroxidopløsning).The methylene chloride extracts are combined and stirred with 9 liters of 3 water, acidifying to a pH of 3 in the aqueous phase (20 cm 6 N hydrochloric acid). The organic phase is separated and washed again with 18 liters of water, adding sodium hydroxide solution to a pH of 9.5 in the aqueous phase (20 cm 6 N sodium hydroxide solution).
20 14199920 141999
Methylenchloridfasen koncentreres under formindsket tryk (5-10 mm Hg) indtil et volumen.på ca. 1 liter.The methylene chloride phase is concentrated under reduced pressure (5-10 mm Hg) to a volume of approx. 1 liter.
Det således fremstillede methylenchloridkoncentrat udhældes langsomt i 10 liter hexan. Opløsningen filtreres. Der fraskilles herved 7,5 g af et inaktivt, uopløseligt stof. Filtratet koncentreres til et volumen på ca. 1 liter. Koncentratet, til hvilket der er sat 1 liter ethylacetat, omrøres i nærværelse af 1 liter vand. Der indstilles til en pH-værdi på 3 ved tilsætning af 6 N saltsyre.The methylene chloride concentrate thus prepared is slowly poured into 10 liters of hexane. The solution is filtered. Thereby, 7.5 g of an inactive, insoluble substance is separated. The filtrate is concentrated to a volume of approx. 1 liter. The concentrate, to which is added 1 liter of ethyl acetate, is stirred in the presence of 1 liter of water. A pH of 3 is adjusted by the addition of 6 N hydrochloric acid.
Den organiske fase fraskilles og koncentreres til et volumen på 1 liter» 30.504 RP udkrystalliserer langsomt ved henstilling af opløsningen ved +4°C. De fremkomne krystaller fraskilles ved filtrering og vaskes med 50 cm^ hexan ved 4°C og tørres derefter ved 35°C under formindsket tryk (5-10 mm Hg). Der fås på denne måde 76 g 30.504 RP som råprodukt på syreform.The organic phase is separated and concentrated to a volume of 1 liter of »30,504 RP slowly crystallizes by recommending the solution at + 4 ° C. The resulting crystals are separated by filtration and washed with 50 cm 2 of hexane at 4 ° C and then dried at 35 ° C under reduced pressure (5-10 mm Hg). In this way, 76 g of 30,504 RP is obtained as crude product in acid form.
c) Rensning: 3 76 g af det ovenfor fremstillede produkt opløses i 1520Jcm acetone. Opløsningen omrøres i 1/2 time i nærværelse af aktivt kul.c) Purification: 3 76 g of the above product is dissolved in 1520 Jcm of acetone. The solution is stirred for 1/2 hour in the presence of activated charcoal.
33
Suspensionen filtreres, og filterkagen vaskes med 100 cm acetone.The suspension is filtered and the filter cake washed with 100 cm of acetone.
33
Filtratet og vaskevæsken forenes. Derefter tilsættes der 850 cm vaiid under langsom omrøring ved 0°C, og blandingen henstilles ved 4°C.The filtrate and washing liquid are combined. Then 850 cm 3 of vydid is added with slow stirring at 0 ° C and the mixture is allowed to stand at 4 ° C.
33
De fremkomne krystaller fraskilles ved filtrering, vaskes med 100 cm af en blanding af acetone og vand i volumenforholdet 1:3 og tørres under formindsket tryk (5-10 mm Hg) ved 35°C. Der fås på denne måde 68,5 g 30.504 RP.The crystals obtained are separated by filtration, washed with 100 cm of a mixture of acetone and water in a volume ratio of 1: 3 and dried under reduced pressure (5-10 mm Hg) at 35 ° C. 68.5 g of 30.504 RP are thus obtained.
d) Omkrystallisation: 68 g af det således fremstillede produkt opløses i 2,8 liter hexan. Opløsningen omrøres i 7 timer, hvorunder produktet langsomt udkrystalliserer.d) Recrystallization: 68 g of the product thus obtained is dissolved in 2.8 liters of hexane. The solution is stirred for 7 hours, during which the product slowly crystallizes.
Suspensionen henstilles i 15 timer ved +4°C, hvorefter krystal- 3 lerne fraskilles ved filtrering, vaskes med 100 cm hexan og tørres under formindsket tryk (5-10 mm Hg) i 12 timer ved 35°C. Der fås på denne måde en første fraktion på 54 g 30.504 RP på syreform.The suspension is left for 15 hours at + 4 ° C, after which the crystals are separated by filtration, washed with 100 cm of hexane and dried under reduced pressure (5-10 mm Hg) for 12 hours at 35 ° C. In this way, a first fraction of 54 g of 30,504 RP is obtained in acid form.
Der kan fremstilles en anden fraktion ved koncentrering af moderluden under formindsket tryk (5-10 mm Hg) til et volumen på 0,4 liter og henstilling i 12 timer ved +4°C, De dannede krystaller 3 fraskilles ved filtrering, vaskes med 20 cm hexan og tørres under formindsket tryk (5-10 mm Hg) i 12 timer ved 35°C. Der fås på denne måde en anden fraktion på 10 g 30.504 RP på syreform.Another fraction can be prepared by concentrating the mother liquor under reduced pressure (5-10 mm Hg) to a volume of 0.4 liters and curing for 12 hours at + 4 ° C. The crystals formed 3 are separated by filtration, washed with 20 hexane and dried under reduced pressure (5-10 mm Hg) for 12 hours at 35 ° C. In this way, a second fraction of 10 g of 30,504 RP is obtained in acid form.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
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DK506577A DK141999B (en) | 1974-03-01 | 1977-11-15 | Process for the preparation of antibiotic substance designated 30,504 RP, its metal salts and its salts with nitrogenous bases. |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
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FR7407066 | 1974-03-01 | ||
FR7407066A FR2262510B1 (en) | 1974-03-01 | 1974-03-01 | |
DK80975AA DK141953B (en) | 1974-03-01 | 1975-02-28 | Growth promoting ruminant feed. |
DK80975 | 1975-02-28 | ||
DK506577 | 1977-11-15 | ||
DK506577A DK141999B (en) | 1974-03-01 | 1977-11-15 | Process for the preparation of antibiotic substance designated 30,504 RP, its metal salts and its salts with nitrogenous bases. |
Publications (3)
Publication Number | Publication Date |
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DK506577A DK506577A (en) | 1977-11-15 |
DK141999B true DK141999B (en) | 1980-08-04 |
DK141999C DK141999C (en) | 1980-12-22 |
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DK506577A DK141999B (en) | 1974-03-01 | 1977-11-15 | Process for the preparation of antibiotic substance designated 30,504 RP, its metal salts and its salts with nitrogenous bases. |
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Country | Link |
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DK (1) | DK141999B (en) |
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1977
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DK506577A (en) | 1977-11-15 |
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