DE3932095C1 - Novel nannocheline and methyl ester(s) - active against Gram positive microorganisms - Google Patents

Novel nannocheline and methyl ester(s) - active against Gram positive microorganisms

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DE3932095C1
DE3932095C1 DE19893932095 DE3932095A DE3932095C1 DE 3932095 C1 DE3932095 C1 DE 3932095C1 DE 19893932095 DE19893932095 DE 19893932095 DE 3932095 A DE3932095 A DE 3932095A DE 3932095 C1 DE3932095 C1 DE 3932095C1
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nannochelin
methanol
nannocheline
ion exchange
exchange resin
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Hans Prof. Dr. Reichenbach
Norbert Dr. Bedorf
Edgar Dr. Forche
Klaus Dr. Gerth
Gerhard Prof. Dr. Hoefle
Herbert Dr. Irschik
Rolf Dr. Jansen
Brigitte Dr. Kunze
Florenz Dr. Sasse
Heinrich Steinmetz
Wolfram Dr. 3300 Braunschweig De Trowitzsch-Kienast
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GESELLSCHAFT fur BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) 3300 BRAUNSCHWEIG DE
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GESELLSCHAFT fur BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) 3300 BRAUNSCHWEIG DE
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
    • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
    • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Nannocheline and its Me esters of formula (I) and their nontoxic salts are new. (In (I), R and R' are each H or Me. Prepn. of cpds. (I) comprises propagation of nannocystis exedens (DSM 5530) in a suitable culture medium in the presence of an anion ion exchange resin; then sepn. of the resin, elution with MeOH, and purification of the active fraction by chromatographic sepn. on sephadex and then silica gel. USE - (I) are new active against Gram positive microorganisms, but incompletely so against Escherichia coli and Saccharomyces cerevisiae.

Description

Der vorliegende Anmeldungsgegenstand betrifft Nannocheline, Verfahren und Mikroorganismus zu deren Herstellung, wie sie in den Ansprüchen definiert sind.The present subject matter relates to Nannocheline, procedures and microorganism for their preparation as described in the Claims are defined.

A. ProduktionsbedingungenA. Production conditions

  • A.1. Produktionsstamm: Das Bakterium Nannocystis exedens, Stamm Na e485, Familie Sorangiaceae, Ordnung Myxobacterales.A.1. Production strain: the bacterium Nannocystis exedens, strain Na e485, Sorangiaceae family, order Myxobacterales.
  • A.2. Herkunft des Produktionsstamms: Isoliert 1985 aus einer Erdprobe, die in South Luangwa, Südafrika, gesammelt worden war.A.2. Origin of the production strain: Isolated in 1985 from one Soil sample collected in South Luangwa, South Africa was.
  • A.3. Beschreibung des Produktionsstamms: Die vegetativen Zellen sind kurze dicke Stäbchen. Auf festen Nährböden wächst der Organismus gut auf Peptonagar, z. B. cy-Agar (Casitone, 0,3%; Hefeextrakt, 0,1%; CaCl₂·2 H₂O 0,1%; Agar 1,5%; pH 7,2) oder auf Hefeagar, z. B. vy/2-Agar (Backhefe 0,5%, bezogen auf Frischgewicht; CaCl₂·2 H₂O 0,1%; Agar 1,5%; pH 7,2). Auf geeigneten Nährböden, z. B. auf Ausstrichen von lebenden Escherichia coli auf Wasseragar (Agar 1,5%; CaCl₂·2 H₂O 0,1%; pH 7,2) bildet der Organismus die arttypischen Fruchtkörper: kleine (Durchmesser 6-110 µm) kugelige oder ovale hellbraune bis schwarzbraune einzeln liegende Sporangiolen. In Flüssigmedien wächst der Stamm in kleinen Zellklümpchen, sowohl in Schüttelkolben bei 160 rpm (100 ml Medium in 250 ml Erlenmeyerkolben bzw. 500 ml Medium in 1000 ml Erlenmeyerkolben), als auch in Bioreaktoren (getestet bis zum 700-l-Maßstab). Als Kulturmedium ist z. B. MDl-Medium geeignet: Pepton aus Casein, tryptisch verdaut, 0,3%; mgSO₄·7 H₂O 0,2%; CaCl₂·2 H₂O 0,05%; pH 7,2. Der Stamm wächst auch gut in Medien auf der Basis technischer Substrate, z. B. Probion PS (Einzellerprotein aus Methylomonas clarae); 1,0%; MgSO₄·7 H₂O 0,1%; pH 7,0. Die Kulturen werden bei 30°C über 3-4 Tage gehalten. Na e485 läßt sich konservieren: z. B. durch Einfrieren vegetativer Zellen von Platten oder Flüssigkulturen in Peptonlösung bei -80°C oder im flüssigen Stickstoff.A.3. Description of the production strain: The vegetative cells are short, thick chopsticks. The grows on solid nutrient media Organism well on peptone agar, e.g. B. cy agar (casitone, 0.3%; Yeast extract, 0.1%; CaCl₂ · 2 H₂O 0.1%; Agar 1.5%; pH 7.2) or on yeast agar, e.g. B. vy / 2 agar (baker's yeast 0.5%, based on fresh weight; CaCl₂ · 2 H₂O 0.1%; Agar 1.5%; pH 7.2). On suitable culture media, e.g. B. on smears of living Escherichia coli on water agar (agar 1.5%; CaCl₂ · 2 H₂O 0.1%; pH 7.2) the organism forms the typical ones Fruiting bodies: small (6-110 µm in diameter) spherical or oval light brown to black brown individually lying sporangioles. The strain grows in liquid media in small lumps of cells, both in shake flasks at 160 rpm (100 ml medium in 250 ml Erlenmeyer flask or 500 ml Medium in 1000 ml Erlenmeyer flasks), as well as in bioreactors (tested up to 700 l scale). As a culture medium is z. B. MDI medium suitable: peptone from casein, tryptic digested, 0.3%; mgSO₄ · 7 H₂O 0.2%; CaCl₂ · 2 H₂O 0.05%; pH 7.2. The trunk also grows well in media based on technical Substrates, e.g. B. Probion PS (unicellular protein from Methylomonas clarae); 1.0%; MgSO₄ · 7 H₂O 0.1%; pH 7.0. The  Cultures are kept at 30 ° C for 3-4 days. Na e485 can be preserved: e.g. B. by freezing vegetative Cells from plates or liquid cultures in peptone solution at -80 ° C or in liquid nitrogen.
  • A.4. Leistungen des Produktionsstamms: In zellfreien Kulturüberständen ist eine eisenkomplexierende Verbindung (Siderophor) nachweisbar, die auch antibiotische Aktivität gegen einige Gram-positive Bakterien zeigt. Die Substanz ist chemisch der Familie der Citrat-Hydroxamate zuzuordnen und wurde Nannochelin genannt.A.4. Services of the production master: In cell-free culture supernatants is an iron complexing compound (siderophore) detectable, which also antibiotic activity against some Gram-positive bacteria. The substance is chemically assigned to the family of citrate hydroxamates and was called Nannochelin.
  • A.5. Die Zugänglichkeit des Stamms: Der Produktionsstamm Na e485 ist bei der deutschen Sammlung von Mikroorganismen (DSM) in Braunschweig als Patentstamm Nr. DSM 5530 hinterlegt.A.5. Accessibility of the tribe: the production strain Na e485 is in the German collection of microorganisms (DSM) in Braunschweig as patent master no. DSM 5530.
  • A.6. Nachweis von Nannochelin: Zum qualitativen Nachweis von Nannochelin werden Kulturüberstände mit Butanol extrahiert und die konzentrierten Extrakte im Agardiffusionstest gegen Brevibacterium ammoniagenes getestet. Alternativ können die Kulturüberstände auch mit XAD gerührt und das XAD nach Absieben mit Methanol eluiert werden. Die Aufarbeitung, Isolierung und Charakterisierung erfolgt gemäß Abschnitt B.A.6. Detection of nannochelin: For the qualitative detection of Nannochelin, culture supernatants are extracted with butanol and the concentrated extracts in the agar diffusion test against Brevibacterium ammoniagenes tested. Alternatively, you can the culture supernatants also stirred with XAD and the XAD after sieving, be eluted with methanol. The processing, Isolation and characterization is done according to section B.
  • A.7. Produktionsbedingungen von Nannochelin: In Schüttelkolben wird Nannochelin in mageren Peptonmedien während des Wachstums gebildet und erreicht am Ende der Wachstumsphase nach ca. 3-4 Tagen die höchste Aktivität.A.7. Nannochelin production conditions: In shake flasks Nannochelin is used in lean peptone media during the Growth formed and reached at the end of the growth phase the highest activity after about 3-4 days.
Fermentationsbeispiel im BioreaktorExample of fermentation in a bioreactor

Bioreaktor (b 50) mit 70 l Inhalt mit Blattrührer. Medium : MDl (Peton aus Casein, tryptisch verdaut 0,3%; MgSO₄·7 H₂O 0,2%; CaCl₂·2 H₂O 0,05%; pH 7,0). 60 l Medium werden mit 5 l einer gut gewachsenen Schüttelkolbenkultur beimpft. Die Belüftungsrate wird auf 200 Nl/h, die Drehzahl auf 200 min-1 eingestellt. Der pO₂-Wert, der zu Beginn der Fermentation 98% Sättigung beträgt, sinkt bis zum Ende der Fermentation nach 90 Stunden kontinuierlich bis auf 70%. Der pH-Wert steigt im Verlauf der Fermentation von 7,0 auf 8,0 an. Bioreactor (b 50) with 70 l content with blade stirrer. Medium: MDl (peton from casein, tryptically digested 0.3%; MgSO₄ · 7 H₂O 0.2%; CaCl₂ · 2 H₂O 0.05%; pH 7.0). 60 l of medium are inoculated with 5 l of a well-grown shake flask culture. The ventilation rate is set to 200 Nl / h, the speed to 200 min -1 . The pO₂ value, which is 98% saturation at the start of the fermentation, drops continuously to 70% by the end of the fermentation after 90 hours. The pH rises from 7.0 to 8.0 in the course of the fermentation.

B. Aufarbeitung, Isolierung und CharakterisierungB. Workup, isolation and characterization Aufarbeitung eines 70-Liter-FermentersRefurbishment of a 70 liter fermenter

Nach Fermentation des Stammes Na e485 in Gegenwart von ca. 0,7 l XAD-Harz wird das Harz abgesiebt und mit 2 l Methanol/Wasser = v/v, danach mit 2 l Methanol eluiert. Die Methanol/Wasser-Phase enthält kein Nannochelin. Dieses wird erst mit der Methanol-Fraktion eluiert. Die Methanol-Fraktion wird bis auf 1 l eingeengt und gegen 2× gegen 250 ml Benzin extrahiert, um von wenig polarem Material abzutrennen.After fermentation of the Na e485 strain in the presence of approx. 0.7 l XAD resin sieved the resin and with 2 l of methanol / water = v / v, then with 2 l of methanol eluted. The methanol / water phase contains no nannochelin. This is only with the Methanol fraction eluted. The methanol fraction is concentrated to 1 l and counter Extracted twice against 250 ml of gasoline to separate from less polar material.

Die Methanol-Fraktion wird eingeengt.The methanol fraction is concentrated.

Der Rohextrakt (ca. 7 g) wird über eine Sephadex®LH-20-Säule (62 mm×63 cm) bei einem Fluß von 4 ml/min mit Methanol in neun Fraktionen fraktioniert. Die ersten beiden Fraktionen (2,2 g) enthalten die Chelatoren. Aus diesen Fraktionen werden im Anschluß über eine preparative Kieselgel RP-18-Säule (479×37 mm) bei Mitteldruck- Betrieb, mit dem Laufmittel Methanol/Triethylammonium-Phosphatpuffer (20 mmol, pH 6,5) = v/v und einem Fluß von 15 ml/min Nannochelin A (1), B (2) und C (3) (Detektion bei λ = 277 nm) erhalten. Zur Entsalzung werden die Fraktionen bei Wasserstrahlvakuum vom Methanol befreit und auf eine XAD-Säule (ca. 200 ml XAD) aufgetragen. Mit zwei Säulenvolumina Wasser wird gewaschen, danach mit zwei Volumina Methanol eluiert. Nach Eindampfen und Trocknen an der Ölpumpe werden 1,1 g Nannochelin A, 0,6 g Nannochelin B und weitere 0,3 g Nannochelin C in Form ihrer Triethylammonium-Salze als zäher Honig erhalten.The crude extract (approx. 7 g) is fractionated on a Sephadex®LH-20 column (62 mm × 63 cm) at a flow of 4 ml / min with methanol in nine fractions. The first two fractions (2.2 g) contain the chelators. These fractions are subsequently used on a preparative silica gel RP-18 column (479 × 37 mm) under medium pressure operation, with the mobile solvent methanol / triethylammonium phosphate buffer (20 mmol, pH 6.5) = v / v and a flow of 15 ml / min nannochelin A (1), B (2) and C (3) (detection at λ = 277 nm). For desalination, the fractions are freed from the methanol under a water jet vacuum and applied to an XAD column (approx. 200 ml XAD). Wash with two column volumes of water, then elute with two volumes of methanol. After evaporation and drying on the oil pump, 1.1 g of nannochelin A, 0.6 g of nannochelin B and a further 0.3 g of nannochelin C are obtained in the form of their triethylammonium salts as viscous honey.

Analytische HPLC für 1 und 2: Laufmittel: Methanol/Phosphatpuffer (0,2 mMol, pH 6,5) = 55/45, Fluß: 1,5 ml, Detektion: Dioden-Array, Säule 12,5 cm×4 mm, HD-Sil-18,5 µm. Retentionszeiten: für 1 R t = 6,0 min, für 2 R t = 2,2 min.Analytical HPLC for 1 and 2: eluent: methanol / phosphate buffer (0.2 mmol, pH 6.5) = 55/45, flow: 1.5 ml, detection: diode array, column 12.5 cm × 4 mm, HD-Sil-18.5 µm. Retention times: for 1 R t = 6.0 min, for 2 R t = 2.2 min.

Analytische HPLC für 2 und 3: Säule, Fluß und Detektion wie oben, Laufmittel: Methanol/Phosphatpuffer = 45/55 (pH 6,5). Für 2 R t = 8,1 min, für 3 R t = 2,0 min.Analytical HPLC for 2 and 3: column, flow and detection as above, eluent: methanol / phosphate buffer = 45/55 (pH 6.5). For 2 R t = 8.1 min, for 3 R t = 2.0 min.

Nannochelin A (1):
UV: λ max (lg ε) = 280 nm (4,4).
¹H-NMR (CD₃OD): δ = 7,62 ppm (6 H, 2×2 H aus Aromat, 2×1 H aus Olefin-Teil), 7,41 (m, 8 H, 2×3 H aus Aromat und 2×1 H aus Olefin-Teil), 4,44 (m, 2×1 H, Lysin- α-H), 3,78 (m, 2×ε-CH₂), 3,78 (m, 4 H, 2×ε-CH₂), 3,74 (s, 6 H, 2×Methylester-CH₃), 3,25 (q, 6 H, TEA-CH₂), 2,8 (m, 4 H, Citronensäure-CH₂), 1,92, 1,75 und 1,49 (m, 12 H, β,γ,δ-Lysin-CH₂), 1,28 (t, 9 H, TEA-CH₃).
¹³C-NMR (CD₃OD): δ = 179,1 ppm (s), 174,1 (2×s), 172,9 (2×s), 172,8 (2×s), 143.5 (2×d), 136,6 (2×s), 130,8 (2×d), 129,9 (4×d), 129,0 (4×d), 118,0 (2×d), 75,8 (s), 53,5 (2×d), 52,6 (2×q), 48,1 (2×t), 47,7 (TEA-t), 45,4 (t), 45,3 (t), 32,3 (t), 32,2 (t), 27,3 (2×t), 23,7 (2×t), 9,1 (TEA-q).
FAB(-)-MS: m/z = 767 (M-H), Hochauflösung: für C₃₈H₄₇N₄O₁₃, gef. 767,3126, ber. 767.3139.
[a] D = -13,1° (c = 0,9 in Methanol).Nannochelin A (1):
UV: λ max (lg ε ) = 280 nm (4.4).
1 H-NMR (CD₃OD): δ = 7.62 ppm (6 H, 2 × 2 H from aromatic, 2 × 1 H from olefin part), 7.41 (m, 8 H, 2 × 3 H from aromatic and 2 × 1 H from olefin part), 4.44 (m, 2 × 1 H, lysine- α -H), 3.78 (m, 2 × ε -CH₂), 3.78 (m, 4 H, 2 × ε -CH₂), 3.74 (s, 6 H, 2 × methyl ester-CH₃), 3.25 (q, 6 H, TEA-CH₂), 2.8 (m, 4 H, citric acid-CH₂) , 1.92, 1.75 and 1.49 (m, 12 H, β, γ, δ -lysine-CH₂), 1.28 (t, 9 H, TEA-CH₃).
13 C-NMR (CD₃OD): δ = 179.1 ppm (s), 174.1 (2 × s), 172.9 (2 × s), 172.8 (2 × s), 143.5 (2 × d) , 136.6 (2 × s), 130.8 (2 × d), 129.9 (4 × d), 129.0 (4 × d), 118.0 (2 × d), 75.8 ( s), 53.5 (2 × d), 52.6 (2 × q), 48.1 (2 × t), 47.7 (TEA-t), 45.4 (t), 45.3 ( t), 32.3 (t), 32.2 (t), 27.3 (2 × t), 23.7 (2 × t), 9.1 (TEA-q).
FAB (-) - MS: m / z = 767 (MH), high resolution: for C₃₈H₄₇N₄O₁₃, found. 767.3126, calc. 767.3139.
[ a ] D = -13.1 ° ( c = 0.9 in methanol).

Nannochelin B (2):
UV: λ max (lg ε) = 280 nm (4,4).
¹H-NMR (CD₃OD): δ = 7,62 ppm (m, 6 H, 2×2 H aus Aromat, 2×1 H aus Olefin-Teil), 7,41 (m, 8 H, 2×3 H aus Aromat, 2×1 aus Olefin), 4,44 (dd, 1 H, α-Lysin-H des Methylester-Teiles), 4,34 (dd, 1 H, α-Lysin-H des Säure-Teiles, 3,78 (m, 4 H, 2×ε-CH₂), 3,74 (s, 3 H, Methylester), 3,21 (q, 12 H, TEA-CH₂), 3,75 (m, 4 H, Citronensäure-CH₂), 1,92, 1,77, 1,51 (m, 12 H, β,γ,δ-Lysin-CH₂), 1,34 (t, 18 H, TEA-CH₃).
¹³C-NMR (CD₃OD): δ = 179,4 ppm (s), 178,6 (s), 174,4 (s), 172,9 (s), 172,2 (s), 168,4 (2×s), 143,5 (2×d), 136,5 (2×s), 130,9 (2×d), 129,9 (4×d), 129,0 (4×d), 117,9 (2 ×d), 75,9 (s), 55,7 (d), 53,5 (d), 52,9 (q), 48,8 (2×t), 47,6 (2×TEA-CH₂), 45,8, 45,7 (2 ×t), 33,4 (t), 32,1 (t), 27,5 (t), 27,2 (t), 23,8 (t), 23,7 (t), 9,2 (3×TEA-CH₃).
[α] D = -5,3 (c = 1,0 in Methanol/Wasser = v/v).
FAB(-)-MS: m/z = 753 (M-H) für C₃₇H₄₅N₄O₁₃.Nannochelin B (2):
UV: λ max (lg ε ) = 280 nm (4.4).
1 H-NMR (CD₃OD): δ = 7.62 ppm (m, 6 H, 2 × 2 H from aromatic, 2 × 1 H from olefin part), 7.41 (m, 8 H, 2 × 3 H from Aromatic, 2 × 1 from olefin), 4.44 (dd, 1 H, α- lysine-H of the methyl ester part), 4.34 (dd, 1 H, α- lysine-H of the acid part, 3, 78 (m, 4 H, 2 × ε -CH₂), 3.74 (s, 3 H, methyl ester), 3.21 (q, 12 H, TEA-CH₂), 3.75 (m, 4 H, citric acid) -CH₂), 1.92, 1.77, 1.51 (m, 12 H, β, γ, δ -lysine-CH₂), 1.34 (t, 18 H, TEA-CH₃).
13 C-NMR (CD₃OD): δ = 179.4 ppm (s), 178.6 (s), 174.4 (s), 172.9 (s), 172.2 (s), 168.4 (2 × s), 143.5 (2 × d), 136.5 (2 × s), 130.9 (2 × d), 129.9 (4 × d), 129.0 (4 × d), 117 , 9 (2 × d), 75.9 (s), 55.7 (d), 53.5 (d), 52.9 (q), 48.8 (2 × t), 47.6 (2nd × TEA-CH₂), 45.8, 45.7 (2 × t), 33.4 (t), 32.1 (t), 27.5 (t), 27.2 (t), 23.8 (t), 23.7 (t), 9.2 (3 x TEA-CH₃).
[ α ] D = -5.3 ( c = 1.0 in methanol / water = v / v).
FAB (-) - MS: m / z = 753 (MH) for C₃₇H₄₅N₄O₁₃.

Nannochelin C (3):
UV: λ max (lg ε) = 280 nm (4,4).
¹³C-NMR (CD₃OD): δ = 179,0 ppm (s), 177,3 (2×s), 172,7 (s), 172,4 (s), 168,3 (2×s), 143,4 (2×d), 136,7 (2×s), 130,8 (2×d), 129,9 (4×d), 129,0 (4×d), 118,1 (2×d), 76,2 (s), 54,9 (2×d), 48,1 (2×t), 47,8 (TEA-t), 45,5 (t), 45,1 (t), 32,8 (2×t), 27,6 (2×t), 24,0 (2×t), 9,2 (TEA-q).
H-NMR (CD₃OD): δ = 7,62 ppm (m, 6 H, 2×2 H aus Aromat, 2×1 H aus Olefin-Teil), 7,41 (m, 8 H, 2×3 H aus Aromat, 2×1 aus Olefin), 4,34 (m, 2 H, 2×α-Lysin-H, 3,78 (m, 4 H, 2×e-CH₂), 3,21 (q, 12 H, TEA-CH₂), 3,75 (m, 4 H, Citronensäure-CH₂), 1,92, 1,77, 1,51 (m, 12 H, β,γ,δ-Lysin-CH₂), 1,34 (t, 18 H, TEA-CH₃).
[a] D = -0,5 (c = 0,5 in Methanol/Wasser = v/v).
FAB(-)-MS: m/z = 739 (M-H) für C₃₆H₄₃N₄O₁₃. Nannochelin C (3):
UV: λ max (lg ε ) = 280 nm (4.4).
13 C-NMR (CD₃OD): δ = 179.0 ppm (s), 177.3 (2 × s), 172.7 (s), 172.4 (s), 168.3 (2 × s), 143 , 4 (2 × d), 136.7 (2 × s), 130.8 (2 × d), 129.9 (4 × d), 129.0 (4 × d), 118.1 (2 × d), 76.2 (s), 54.9 (2 × d), 48.1 (2 × t), 47.8 (TEA-t), 45.5 (t), 45.1 (t) , 32.8 (2 × t), 27.6 (2 × t), 24.0 (2 × t), 9.2 (TEA-q).
H-NMR (CD₃OD): δ = 7.62 ppm (m, 6 H, 2 × 2 H from aromatic, 2 × 1 H from olefin part), 7.41 (m, 8 H, 2 × 3 H from Aromatic, 2 × 1 from olefin), 4.34 (m, 2 H, 2 × α- lysine-H, 3.78 (m, 4 H, 2 × e -CH₂), 3.21 (q, 12 H , TEA-CH₂), 3.75 (m, 4 H, citric acid-CH₂), 1.92, 1.77, 1.51 (m, 12 H, β, γ, δ -lysine-CH₂), 1, 34 (t, 18H, TEA-CH₃).
[ a ] D = -0.5 ( c = 0.5 in methanol / water = v / v).
FAB (-) - MS: m / z = 739 (MH) for C₃₆H₄₃N₄O₁₃.

C. Biologische Charakterisierung von NannochelinC. Biological characterization of nannochelin

Die antibiotische Aktivität wird mit Hilfe des Agardiffusionstests anhand des Hemmhofdurchmessers bzw. im Verdünnungsreihentest aus der Kulturdichte ermittelt. Nannochelin hemmt das Wachstum einiger Gram-positiver Bakterien, sowie inkomplett auch das Wachstum von Saccharomyces cerevisiae und E. coli.The antibiotic activity is checked using the agar diffusion test based on the inhibitor diameter or in Dilution series test determined from the culture density. Nannochelin inhibits the growth of some Gram-positive Bacteria, as well as incomplete the growth of Saccharomyces cerevisiae and E. coli.

Die MHK für Brevibacterium ammoniagenes beträgt 1,5 µg/ml und für Staphylococcus aureus 25 µg/ml.The MIC for Brevibacterium ammoniagenes is 1.5 µg / ml and for Staphylococcus aureus 25 µg / ml.

Claims (4)

1. Nannocheline der allgemeinen Formel in der entweder
R′ = Me und R′′ = Me oder
R′ = Me und R′′ = H oder
R′ = H und R′′ = H bedeuten,
sowie ihre therapeutisch verträglichen Salze.1. Nannocheline of the general formula in either
R ′ = Me and R ′ ′ = Me or
R ′ = Me and R ′ ′ = H or
R ′ = H and R ′ ′ = H mean
as well as their therapeutically acceptable salts. 2. Verfahren zur Herstellung von Nannochelinen nach Anspruch 1, dadurch gekennzeichnet, daß man
  • - Nannocystis exedens DSM 5530 in einem Kohlenstoff- und Stickstoffquellen sowie Mineralsalze enthaltenden flüssigen Medium in Gegenwart eines Ionenaustauscherharzes fermentiert,
  • - das Ionenaustauscherharz abtrennt und eluiert,
  • - das Eluat an einer Sephadex®-Säule fraktioniert,
  • - die eisenkomplexierende Aktivität zeigenden Fraktionen an einer Umkehrphasen-Kieselgelsäule in Fraktionen mit
    • a) Nannochelin A
      (R t = 6,0 min; Säule: 12,5 cm×4 mm, HD-Sil-18, 5 µm; Fluß: 1,5 ml; Laufmittel: Methanol/Phosphatpuffer (0,2 mMol, pH 6,5); Detektion: Dioden Array,
    • b) Nannoochelin B
      (R t = 2,2 min; Bedingungen wie bei Nannochelin A) und
    • c) Nannochelin C
      (R t = 2,0 min; Bedingungen wie bei Nannochelin A, außer Laufmittel: Methanol/Phosphatpuffer [45/55, pH 6,5])
  • auftrennt und gegebenenfalls Nannochelin A, B und/oder C isoliert.
2. A process for the preparation of nannochelins according to claim 1, characterized in that
  • - Nannocystis exedens DSM 5530 is fermented in a liquid medium containing carbon and nitrogen sources as well as mineral salts in the presence of an ion exchange resin,
  • - the ion exchange resin is separated and eluted,
  • - the eluate is fractionated on a Sephadex® column,
  • - The fractions showing the iron-complexing activity on a reversed-phase silica gel column in fractions
    • a) Nannochelin A
      ( R t = 6.0 min; column: 12.5 cm × 4 mm, HD-Sil-18, 5 µm; flow: 1.5 ml; eluent: methanol / phosphate buffer (0.2 mmol, pH 6.5 ); Detection: diode array,
    • b) Nannoochelin B
      ( R t = 2.2 min; conditions as for Nannochelin A) and
    • c) Nannochelin C
      ( R t = 2.0 min; conditions as for Nannochelin A, except eluent: methanol / phosphate buffer [45/55, pH 6.5])
  • separates and optionally isolated Nannochelin A, B and / or C.
3. Verfahren nach Anspruch 2, dadurch gekennzeichnet, daß man das Ionenaustauscherharz mit Methanol eluiert.3. The method according to claim 2, characterized in that one the ion exchange resin eluted with methanol. 4. Nannocystis exedens DSM 5530 zur Durchführung des Verfahrens gemäß Anspruch 2.4. Nannocystis exedens DSM 5530 to perform the procedure according to claim 2.
DE19893932095 1989-09-26 1989-09-26 Novel nannocheline and methyl ester(s) - active against Gram positive microorganisms Expired - Fee Related DE3932095C1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110306563A1 (en) * 2008-12-17 2011-12-15 Sanofi Macrolactone derivatives, method for the production thereof and use thereof for the treatment of cancer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NICHTS ERMITTELT *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110306563A1 (en) * 2008-12-17 2011-12-15 Sanofi Macrolactone derivatives, method for the production thereof and use thereof for the treatment of cancer

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