Antibiotic R2, a new angucyclinone compound from Streptosporangium sp. Sg

A Kawashima

A Zitouni

H Boudjella

J Rohr

K Krohn

M Tsunakawa

N Sabaou

S Kondo

T Tsuno

Y Matsuda

Y Tsurumi

International audienceAngucyclinones are a subclass of angucyclines, which are defined as microbial quinone natural products related to tetracyclines and anthracyclines and bearing, as characteristic structural feature, a tetracyclic benz[a]anthracene ring system assembled in an angular manner. This structural moiety is biosynthetically derived from a decaketide chain formed via the polyketide biosynthetic pathway. The term ‘angucycline’ includes molecules with hydrolysable sugar moieties, whereas ‘angucyclinone’ refers to a sugarless compound or a compound with a C-glycosidic linked sugar moiety.  Angucyclines are isolated from the fermentation broth of actinomycetes, mostly from the genus Streptomyces, but also from Actinomadura, Nocardia and Streptosporangium genera.1 Angucyclines show a broad spectrum of biological activities including antitumor, antibacterial, antifungal, antiviral, enzyme inhibitory and platelet aggregation inhibitory properties. In a continuous search for new bioactive compounds from actinomycetes other than the genus Streptomyces, several Saharan soil samples collected in arid ecosystems were explored. Among the isolates, we were interested by a new actinomycete strain belonging to the genus Streptosporangium and producing a new compound (R2) identified as a new angucyclinone. This paper describes the isolation, structure elucidation and antimicrobial activities of compound R2

Boudjella, Hadjira

Zitouni, Abdelghani

Coppel, Yannick

Mathieu, Florence

Monje, Marie-Carmen

Sabaou, Nasserdine

Lebrihi, Ahmed

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Antibiotic R2, a new angucyclinone compound from Streptosporangium sp. Sg3

Hadjira Boudjella

Abdelghani Zitouni

Yannick Coppel

Florence Mathieu

Marie-Carmen Monje

Nasserdine Sabaou

Ahmed Lebrihi

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The Journal of Antibiotics

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OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible Any correspondence concerning this service should be sent  to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr This is an author’s version published in: http://oatao.univ-toulouse.fr/23172To cite this version: Boudjella, Hadjira and Zitouni, Abdelghani and Coppel, Yannick and Mathieu, Florence and Monje, Marie-Carmen and Sabaou, Nasserdine and Lebrihi, Ahmed Antibiotic R2, a new angucyclinone compound from Streptosporangium sp. Sg3. (2010) The Journal of Antibiotics, 63 (12). 709-711. ISSN 0021-8820Official URL: https://doi.org/10.1038/ja.2010.111NOTEAntibiotic R2, a new angucyclinone compoundfrom Streptosporangium sp. Sg3Hadjira Boudjella1, Abdelghani Zitouni1, Yannick Coppel2, Florence Mathieu3, Marie-Carmen Monje3, Nasserdine Sabaou1 and Ahmed Lebrihi3Keywords: actinomycete; new angucyclinone; Streptosporangium; structure elucidationAngucyclinones are a subclass of angucyclines, which are defined asmicrobial quinone natural products related to tetracyclines andanthracyclines and bearing, as characteristic structural feature, atetracyclic benz[a]anthracene ring system assembled in an angularmanner.1 This structural moiety is biosynthetically derived from adecaketide chain formed via the polyketide biosynthetic pathway.The term ‘angucycline’ includes molecules with hydrolysable sugarmoieties, whereas ‘angucyclinone’ refers to a sugarless compound or acompound with a C-glycosidic linked sugar moiety.1,2 Angucyclinesare isolated from the fermentation broth of actinomycetes, mostlyfrom the genus Streptomyces, but also from Actinomadura, Nocardiaand Streptosporangium genera.1 Angucyclines show a broad spectrumof biological activities including antitumor,1,2 antibacterial, antifungal,antiviral,3,4 enzyme inhibitory5,6 and platelet aggregation inhibitoryproperties.7,8 In a continuous search for new bioactive compoundsfrom actinomycetes other than the genus Streptomyces, several Saharansoil samples collected in arid ecosystems were explored.9–11 Amongthe isolates, we were interested by a new actinomycete strain belongingto the genus Streptosporangium and producing a new compound (R2)identified as a new angucyclinone. This paper describes the isolation,structure elucidation and antimicrobial activities of compound R2.Details on the isolation and the taxonomy of the producingorganism Streptosporangium sp. Sg3 were described in a previouspaper.11 To isolate compound R2, Streptosporangium sp. Sg3 wascultivated at 30 1C for 9 days in Erlenmeyer flasks (500ml) containing100ml of ISP2 broth (yeast extract 4 g, malt extract 10 g, glucose 4 g,in 1 l distilled water, pH 7.2) on a rotary shaker (250 r.p.m.). Thecultures (8 l) were centrifuged and filtered to remove mycelium. Theculture filtrate was extracted with an equal volume of n-butanol togenerate a crude extract (1.9 g). The latter was fractionated by sizeexclusion chromatography on Sephadex LH-20 (75%MeOH in H2O),resulting in five fractions, I–V. Fraction IV including R2, was ofred color and exhibited an antibacterial activity. It was subjectedto semipreparative reversed-phase HPLC using an InterchimUP5ODB column (2507.8mm) (Interchim, Montluc¸on, France)and developed using a continuous grade from 20 to 75% MeOH inH2O (UV detection at 220 nm), yielding three active fractions (1, 2, 3).The major peak 2 with the main antibacterial activity was subjected torepeated HPLC to yield 3.5mg of compound R2.Compound R2 was obtained as a red powder (optical rotation[a]D21 +56 (c 0.11, MeOH). The ESI-MS spectrum contained an ionpeak atm/z 475.1 [M–H], and its molecular formula was determinedby HRESI-MS analysis as C26H19O9 (calcd 475.43 for (M–H), found475.43). The UV absorption maximum at 535 nm was because of thered color and suggested a quinone chromophore. Absorptions at 3259,2949 and 1721 cm1 in the IR spectrum of R2 were characteristic ofhydroxy, methyl and carbonyl groups, respectively. R2 was soluble inMeOH and DMSO, and insoluble in chloroform, n-hexane and H2O.The structure of R2 (Figure 1) was determined by 1H and 13CNMR spectroscopy and by using 1H–1H COSY45, 1H–13C HMQCand 1H–13C HMBC experiments. The 1H and 13C chemical shifts ofcompound R2 are given in Table 1. The 13C and Heteronuclear Single-Quantum Correlation spectra showed 26 carbon signals with a largenumber of quaternary carbons (17 out of 26). From the 13C data, itwas possible to discern two keto-carbonyl groups (dc 188.4 and 186.5),one carboxylic acid group (dc 174.1), 19 sp2-hybridized carbons (dcfrom 160.2 to 114.7), two sp3-hybridized carbons bearing an electro-negative heteroatom (dc 73.0 and 72.5), one sp3-hybridized carbon (dc22.6) and two methyl groups (dc 22.4 and 12.4). In CD3OD, the 1HNMR spectrum revealed two ortho-coupled aromatic protons (dH7.82 and 7.66, 2H, J¼7.5Hz), two aromatic protons (dH 8.24 and7.02, 2H, s), an AB system (dH 4.45 and 4.36, JAB¼10.7Hz), an ethylgroup (dH 2.83 and 1.32, 5H, J¼7.4Hz) and a methyl group (dH 2.70,3H, s). In DMSO, NMR signals were significantly broader but the1Laboratoire de Recherche sur les Produits Bioactifs et la Valorisation de la Biomasse, Ecole Normale Supe´ rieure de Kouba, Alger, Alge´ ria; 2CNRS, LCC (Laboratoire de Chimie de Coordination), Toulouse, France and 3De´ partement ‘Bioproce´ de´ s & Syste` mes Microbiens’, ENSAT/INPT, Universite´ de Toulouse, Laboratoire de Ge´ nie Chimique, UMR 5503 (CNRS/INPT/UPS), Castanet-Tolosan, FranceCorrespondence: Professor A Lebrihi, De´ partement of ‘Bioproce´ de´ s & Syste` mes microbiens’, ENSAT/INPT, Universite´ de Toulouse, Laboratoire de Ge´ nie Chimique, UMR 5503(CNRS/INPT/UPS), 1 avenue de l’Agrobiopole, B.P. 32 607, Auzeville-Tolosane, Castanet-Tolosan 31 326, France.E-mail: lebrihi@ensat.fr1H NMR spectrum showed additional resonances: four enol orcarboxylic acid protons (dH 17.64, 13.98, 13.20 and 13.08, 4H, s)and two hydroxyl signals (dH 5.92 and 5.69, 2H, s). The 2D 1H–1H and1H–13C experiments and especially the long range 1H–13C couplingsobserved in the HMBC spectrum permitted to establish the presence ofa benzo[a]naphtacenequinone skeleton. Only four carbons did notshow any correlation in the HMBC spectrum (C1, C7a, C14 and C15).Their assignment has been confirmed by comparison with NMR dataof the pradimicin antibiotic family.12 The peak of the four enol orcarboxylic acid protons was too broad to give HMBC correlations andso could not be exactly assigned. The observed coupling constant of10.7Hz between H5 and H6 suggested that the dihedral angles betweenH5 and H6 are near 1801 and is consistent with a trans-diaxalorientation. This value is similar with ones found for related com-pounds3,12 where a 5S,6S absolute configuration has been deduced.The structure of R2 was determined to be a benzo[a]naphtacene-2-carboxylic acid,4,7,11,12-tetrahydro-1,5,6,9,14-pentahydroxy-8,13-dioxo-3-methyl-10-ethyl.The minimum inhibitory concentrations (MICs) of R2 weredetermined by a conventional agar dilution method using nutrientagar medium. Compound R2 showed strong activity against theGram-positive bacteria Micrococcus luteus ATCC 9314 and Bacillussubtilis ATCC 6633 (MICs¼0.5 and 1mgml1, respectively), andmoderate activity against Staphylococcus aureus CIP 7625(MIC¼10mgml1). A weak activity was observed against Listeriamonocytogenes CIP 82110 and Mycobacterium smegmatis ATCC 607(MICs¼40 and 50mgml1, respectively). Compound R2 showed noactivity (MICs4100mgml1) against the Gram-negative bacteria(Escherichia coli ATCC 10536 and Pseudomonas fluorescens number412), the fungi (Mucor ramannianus NRRL 1829 and Aspergilluscarbonarius number M333) and the yeasts (Saccharomyces cerevisiaeATCC 4226 and Candida albicans number 224).In conclusion, from the results presented in this paper, we foundthat strain Streptosporangium sp. Sg3 produces a new angucyclinonethat displayed antibacterial activities.ACKNOWLEDGEMENTSA part of this work was supported by the grant from ‘Comite´ d’Evaluationet de Prospective de Coope´ration Inter-universitaire Franco-Alge´rienne’(Programme Hubert Curien Tassili).1 Rohr, J. & Thiericke, R. Angucycline group antibiotics. Nat. Prod. Rep. 9, 103–137(1992).2 Krohn, K. & Rohr, J. Angucyclines: total syntheses, new structures, and biosyntheticstudies of an emerging new class of antibiotics. Top. Curr. Chem. 188, 128–195(1997).3 Tsunakawa, M. et al. The structures of pradimicins A, B, and C: a novel family ofantifungal antibiotics. J. Org. Chem. 54, 2532–2536 (1989).4 Kondo, S., Gomi, S., Ikeda, D., Hamada, M. & Takeuchi, T. Antifungal andantiviral activities of benanomycins and their analogues. J. Antibiot. 44, 1228–1236(1991).ODAOHOH OHOHOOOH1617183615814a121OHB 5913COOOHHHHOHHHOHOHHMeHOOHO1H-13C HMBC correlations1H-1H COSY correlationsFigure 1 Structure of angucyclinone R2 (a), and HMBC and COSY correlations (b).Table 1 1H and 13C NMR data assignments of CD3OD at 298KPosition dC, mult. dH (J in Hz)1 160.2, qCa —2 118.5, qC —3 143.1, qC —4 116.9, CH 7.02,s4a 147.4, qC —5 72.5, CH 4.36, d (10.7)6 73.0, CH 4.45, d (10.7)6a 141.9, qC —7 114.7, CH 8.24, s7a 131.6, qC —8 188.4, qC —8a 115.2 qC —9 160.1, qC —10 139.9, qC —11 135.3, CH 7.66, d (7.5)12 118.7, CH 7.82, d (7.5)12a 132.0, qC —13 186.5, qC —13a 117.4, qC —14 158.6, qCa —14a 128.6, qC —14b 114.9, qC —15 174.1, qC —16 22.4, CH3 2.70, s17 22.6, CH2 2.83, q (7.4)18 12.4, CH3 1.32, t (7.4)OH-1b,c — 17.64, sOH-5c — 5.69, sOH-6c — 5.92, sOH-9b,c — 13.98, sOH-14b,c — 13.08, sCOOHb,c — 13.20, sa,bResonance assignment may be interchangeable.cIn DMSO-d6.Mult. stands for multiplicity. qC, CH, CH2 and CH3 indicate respectively quaternary, tertiary,secondary and primary carbons.5 Tsurumi, Y. et al. WS79089A, B and C, new endothelin converting enzyme inhibitorsisolated from Streptosporangium roseum No. 79089. Taxonomy, fermentation, isolation,physico-chemical properties and biological activities. J. Antibiot. 47, 619–630 (1994).6 Matsuda, Y. & Kase, H. KS-619-1, a new inhibitor of Ca2+ and calmodulin-dependentcyclic nucleotide phosphodiesterase from Streptomyces californicus. J. Antibiot. 40,1104–1110 (1987).7 Kawashima, A., Kishimura, Y., Tamai, M. & Hanada, K. New platelet aggregationinhibitors. Chem. Pharm. Bull. 37, 3429–3431 (1989).8 Kawashima, A. et al. PI-083, a new platelet aggregation inhibitor. J. Antibiot. 41,1913–1914 (1988).9 Sabaou, N. et al. Les sols des oasis du Sahara alge´rien, source d’actinomyce`tes raresproducteurs d’antibiotiques. Se´cheresse 9, 147–153 (1998).10 Zitouni, A. et al. Saccharothrix algeriensis sp. nov., isolated from Saharan soil. Int. J.Syst. Evol. Microbiol. 54, 1377–1381 (2004).11 Boudjella, H. et al. Isolation and partial characterization of pigment-like antibioticsproduced by a new strain of Streptosporangium isolated from an Algerian soil. J. Appl.Microbiol. 103, 228–236 (2007).12 Tsuno, T. et al. Biosynthesis of the pradimicin family of antibiotics. II. Fermentation,isolation and structure determination of metabolites associated with the pradimicinsbiosynthesis. J. Antibiot. 46, 420–429 (1993).

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Antibiotic R2, a new angucyclinone compound from Streptosporangium sp. Sg3

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