Intern
    Prof. Dr. G. Bringmann

    New Bioactive Compounds from Marine Organisms

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    1. Key Words:

    Pharmacologically active natural secondary metabolites from marine organisms; bioassay-guided search for new potent agents; spectroscopy-guided search for novel-type structures using hyphenated techniques such as HPLC coupled to MS/MS, NMR, and CD (see also 'Natural Product Analysis'); isolation, structural elucidation; determination of full absolute stereostructures by quantum chemical CD calculations (see also ‘Computational Chemistry’); elucidation of biosynthetic pathways; chemical modification; partial or total synthesis; collaborative research network BIOTECmarin (see also www.biotecmarin.de).

    2. Graphical Abstract:

    Subtopic A:

    Sorbicillactone A, the First Sorbicillinoid Alkaloid from a Sponge-Derived Penicillium chrysogenum Strain, Exhibiting Antileukemic Activity

     

    Figure 1: Identification, structrual elucidation, and biosynthetic origin of sorbicillactone A, the first member of the novel class of sorbicillin-derived alkaloids, isolated from a strain of Penicillium chrysogenum from a specimen of the Mediterranean sponge Ircinia fasciculata. Sorbicillactone A is a potential new lead structure exhibiting selective antileukemic, anti-HIV, and neuroprotective activities.
    Figure 2: Biotechnological production and isolation of pure sorbicillactone A in five steps on a large scale for ongoing preclinical studies.
    Figure 3: Structures of further metabolites isolated from the sorbicillactones producing Penicillum chrysogenum strain.

    Subtopic B:

    The LC-MS/MS-NMR-CD Triad in Marine Natural Products Chemistry:

    Discovery of Secondary Metabolites from Sponge-Derived Fungi

     

    Figure 4: The analytical hyphenation triad HPLC-MS/MS-NMR-CD and its application to the online structural elucidation of marine natural products from sponge-derived fungi.

    3. Brief Description:

    Our research network, the Center of Excellence BIOTECmarin, has embarked on the search for new bioactive compounds from marine sponges, fungi or bacteria, as well as their further development as potential novel drugs (see Flow Chart). The chemical structures of the secondary metabolites synthesized by sponges or by their associated microorganisms are highly diverse, and thus already of relevance per se. The main interest in these compounds, however, results from their frequently strong and often highly specific bioactivities.

    The discovery of the antileukemic alkaloid sorbicillactone A (see Figure 1) and its dihydro analog sorbicillactone B (see Figure 2) represents an illustrative example of the fruitful cooperation within BIOTECmarin. These two compounds were isolated from a strain of Penicillium chrysogenum cultured from a sample of the Mediterranean sponge Ircinia fasciculata [1-3]. They possess a unique bicyclic lactone structure, seemingly derived from sorbicillin and alanine. Among the numerous known sorbicillin-derived structures, they are the first sorbicillinoid natural products found to contain nitrogen, and thus the first representatives of a novel-type of 'sorbicillin alkaloids'. Sorbicillactone A and B originate via a likewise unprecedented pathway elucidated in our laboratories by applying feeding experiments with 13C-labelled precursors (see Figure 1). Our investigations clearly demonstrate the carbon skeleton of the sorbicillactones to be derived from a 'sorbicillinol unit' formed from acetate and S-adenosyl-methionine, the amino acid alanine, and a biosynthetic equivalent of fumaric acid [2].

    Sorbicillactone A exhibits promising activities in several mammalian, viral, and neuronal test systems [1-4], in particular a highly selective cytostatic activity against murine leukemic lymphoblasts (L5178y), the ability to protect human T cells against the cytopathic effects of HIV-1, and it is capable to act as a neuroprotective agent in primary neurons (see Figure 1). Despite its almost identical molecular structure, sorbicillactone B is significantly less active than sorbicillactone A (by a factor of 10). For the evaluation of the therapeutical potential of the more promising sorbicillactone A, for preclinical investigations, and for SAR studies on semisynthetic analogs, we developed an efficient method for its large-scale production, isolation and purification (see Figure 2) [5,6]. The procedure includes the following steps: (1) fermentation of the fungus in static surface cultures under optimized conditions (pH, temperature, medium), (2) extraction of the culture broth using a nonionic resin (XAD-16), followed by elution with methanol; (3) purification of the crude extract with ethyl acetate at various pH values; (4) isolation of crude sorbicillactone A by fast centrifugal partitioning chromatography (FCPC); and (5) isolation of pure sorbicillactone A by gel permeation chromatography on Sephadex LH-20 with methanol as the eluent achieving a successful elimination of the less active, chromatographically almost identical sorbicillactone B. By this method, more than 100 g of pure sorbicillactone A have meanwhile been isolated for ongoing pharmacological and toxicological studies.

    Furthermore, saltwater cultures of this Penicillium chrysogenum strain yielded the known sorbicillinoid fungal metabolites oxosorbicillinol, sorbicillin, and bisvertinolone, as well as the alkaloids meleagrine and roquefortine C (see Figure 3) [1,2].

    The application of hyphenated techniques like high-performance liquid chromatography (HPLC) coupled to nuclear magnetic resonance spectroscopy (NMR), to electrospray ionization tandem mass spectrometry (ESI-MS/MS), and to circular dichroism spectroscopy (CD) is a sensitive and powerful tool in natural product chemistry permitting a fast screening of the metabolic profiles of marine organisms (see Figure 4), with a minimum amount of material. By the combined application of HPLC-NMR, HPLC-ESI-MS/MS, and HPLC-CD, it is not only possible to rapidly identify known structures such as cyclopiazonic acid, but also to investigate new metabolites and to establish their full absolute stereostructures online, directly from crude extracts, without the necessity of first isolating the compounds (see also 'Natural Product Analysis') [7-12]. For example, from a strain of the fungus Emericella variecolor derived from the marine sponge Haliclona valliculata, the novel natural product isoemericellin was structurally elucidated after HPLC-UV, -MS, and -NMR studies of the extract. Moreover, this fungus was found to produce the known metabolites stromemycin and shamixanthone (see Figure 4) [8]. Petrosifungin A, a previously unknown cyclodepsipeptide was detected in a strain of Penicillium brevicompactum derived from a specimen of the Mediterranean sponge Petrosia ficiformis, besides the known fungal metabolite brevianamide (see Figure 4) [7,9]. The LC-MS/MS-NMR-CD online analysis of Penicillium cf. montanense isolated from the sponge Xestospongia exigua led to the identification and configurational assignment of novel macrocyclic lactones like xestodecalactone A (see Figure 4) [7,10]. The absolute configuration of xestodecalactone A was established by quantum chemical CD calculations (see also 'Computational Chemistry') and a stereochemically unambiguous first total synthesis [11]. Furthermore, daminin (see Figure 4) from the Mediterranean sponge Axinella damicornis, one of the relatively few non-brominated pyrrole alkaloids of marine origin showing neuroprotective properties, was structurally investigated and a short and efficient total synthesis was elaborated [12].

     

    4. Selected Publications:

    [1]

    G. Bringmann, G. Lang, J. Mühlbacher, K. Schaumann, S. Steffens, P.G. Rytik, U. Hentschel, J. Morschhäuser, W.E.G. Müller; Sorbicillactone A: a structurally unprecedented bioactive novel-type alkaloid from a sponge-derived fungus. In: Marine Molecular Biotechnology (W.E.G. Müller, ed.), Springer-Verlag, Berlin, Heidelberg, 2003, pp. 231-253.

    [2]

    G. Bringmann, G. Lang, T.A.M. Gulder, H. Tsuruta, J. Mühlbacher, K. Maksimenka, S. Steffens, K. Schaumann, R. Stöhr, J. Wiese, J.F. Imhoff, S. Perović-Ottstadt, O. Boreiko, W.E.G. Müller; The first sorbicillinoid alkaloids, the antileukemic sorbicillactones A and B, from a sponge-derived Penicillium chrysogenum strain. Tetrahedron 2005, 61, 7252-7265.

    [3]

    G. Bringmann, D. Wunderlich; Sorbicillacton A – eine chemische Schnitzeljagd. In: Vorbild Natur – Stand und Perspektiven der Naturstoff-Forschung in Deutschland. DECHEMA e.V., Frankfurt am Main, 2007, pp. 36-43.

    [4]

    G. Bringmann, G. Lang, J. Mühlbacher, K. Schaumann, S. Steffens, W.E.G. Müller (Johannes-Gutenberg-Universität, Mainz, DE; Freistaat Bayern vertreten druch die Julius-Maximilians-Universität, Würzburg, DE); Sorbicillacton-A-Derivate zur Behandlung von Tumor- und Viruserkrankungen. Europäische Patentschrift EP 1 532 129 B1 (22.11.2006). Internationale Veröffentlichungsnummer: WO 2004/026854 (01.04.2004).

    [5]

    G. Bringmann, T.A.M. Gulder, G. Lang, S. Schmitt, R. Stöhr, J. Wiese, K. Nagel, J.F. Imhoff; Large-scale biotechnological production of the antileukemic marine natural product sorbicillactone A. Marine Drugs 2007, 5, 23-30.

    [6]

    G. Bringmann, G. Lang, T.A.M. Gulder, K. Schaumann, W.E.G. Müller, S. Perović-Ottstadt, R. Stöhr, J. Wiese, R. Schmaljohann, J.F. Imhoff (Johannes-Gutenberg-Universität, Mainz, DE; Julius-Maximilians-Universität, Würzburg, DE); Verfahren zur Herstellung von Sorbicillacton A. Deutsche Patentschrift DE 10 2004 004 901 B4 (05.01.2006).

    [7]

    G. Bringmann, G. Lang; Full absolute stereostructures of natural products directly from crude extracts: the HPLC-MS/MS-NMR-CD 'triad'. In: Marine Molecular Biotechnology (W.E.G. Müller, ed.), Springer-Verlag, Berlin, Heidelberg, 2003, pp. 89-116.

    [8]

    G. Bringmann, G. Lang, S. Steffens, E. Günther, K. Schaumann; Evariquinone, isomericellin, and stromemycin from a sponge-derived strain of the fungus Emericella variecolor. Phytochemistry 2003, 63, 437-443.

    [9]

    G. Bringmann, G. Lang, S. Steffens, K. Schaumann; Petrosifungins A and B, novel cyclodepsipeptides from a sponges-derived strain of Penicillium brevicompactum. J. Nat. Prod. 2004, 67, 311-315.

    [10]

    R.A. Edrada, M. Heubes, G. Brauers, V. Wray, A. Berg, U. Gräfe, M. Wohlfarth, J. Mühlbacher, K. Schaumann, Sudarsono, G. Bringmann, P. Proksch; Online analysis of xestodecalactones A-C, novel bioactive metabolites from the fungus Penicillium cf. montanense and their subsequent isolation from the sponge Xestospongia exigua. J. Nat. Prod. 2002, 65, 1598-1604.

    [11]

    G. Bringmann, G. Lang, M. Michel, M. Heubes; Stereochemical assignment of the fungal metabolite xestodecalactone A by total synthesis. Tetrahedron Lett. 2004, 45, 2829-2831.

    [12]

    A. Aiello, M. D'Esposito, E. Fattorusso, M. Menna, W.E.G. Müller, S. Perović-Ottstadt, H. Tsuruta, T.A.M. Gulder, G. Bringmann; Daminin, a bioactive pyrrole alkaloid from the Mediterranean sponge Axinella damicornis. Tetrahedron 2005, 61, 7266-7270.

    5. Cooperations and Research Grants:

    Within the special research project entitled „BIOTECmarin – Center of Competence: Molecular Biotechnology and New Agents from Marine Sponges and Associated Microorganisms“, sponsored by the Bundesminsterium für Bildung, Forschung, Wissenschaft und Technologie (BMBF), in collaboration with

    (a) Prof. Dr. W.E.G. Müller, Institut für Physiologische Chemie, Abteilung für Angewandte Molekularbiologie, Johannes-Gutenberg-Universität Mainz;

    (b) Dr. K. Schaumann, Alfred-Wegener-Institut für Polar- und Meeresforschung, Bremerhaven;

    (c) Prof. Dr. J.F. Imhoff, Marine Mikrobiologie, Leibniz-Institut für Meereswissenschaften, IFM-GEOMAR, Kiel;

    (d) Prof. Dr. P. Proksch, Institut für Pharmazeutische Biologie, Heinrich-Heine-Universität Düsseldorf;

    (e) PD Dr. U. Hentschel, Zentrum für Infektionsforschung, Julius-Maximilians-Universität Würzburg;

    (f) Prof. Dr. J. Piel, Kekulé-Institut für Organische Chemie und Biochemie; Rheinische Friedrich-Wilhelms-Universität Bonn.

     

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