5-Hydroxyramulosin, a New Natural Product Produced by Phoma tropica, a Marine-Derived Fungus Isolated from the Alga Fucus spiralis

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The fungus Phoma tropica was isolated from the inner tissue of the marine brown alga Fucus spiralis. After large-scale cultivation the fungus was investigated for its secondary metabolite content and found to contain the new natural product 5-hydroxyramulosin (1) together with 7-methoxycoumarin. Both structures were elucidated using spectroscopic methods, mainly 1D and 2D NMR, and in the case of 1, a single crystal X-ray diffraction analysis.

In recent years marine fungi have been shown to be an increasingly interesting and potentially valuable source of biologically active novel natural products [1], [2], [3]. The fungal genus Phoma is known for its diverse spectrum of secondary metabolites as reflected by the numerous studies into its secondary metabolite chemistry e. g., the antibiotic phomalide which was isolated from Phoma lingam [4], cytochalasin T produced by P. exigua var. heteromorpha [5], and betaenone D found in P. betae [6]. A chemotaxonomic investigation of this genus showed that marine-derived Phoma spp. differ significantly from their terrestrial counterparts with respect to their secondary metabolite content [7]. A preliminary investigation into the biological activity of the EtOAc extract of a marine-derived fungal strain of P. tropica, obtained from the marine brown alga Fucus spiralis, was undertaken and showed this fungus to produce antimicrobially active compounds.

In the present study the isolation and structure elucidation of 5-hydroxyramulosin (1) and 7-methoxycoumarin (2) obtained from P. tropica are described. The fungus was cultivated on 14 L of a liquid malt soya medium with added artificial sea salt. Successive fractionation of the EtOAc extract by vacuum-liquid chromatography (VLC), and normal (Si-60) and reversed (RP-18) phase HPLC yielded compounds 1 and 2.

5-Hydroxyramulosin (1) has the molecular formula C₁₀H₁₄O₄, as deduced by accurate mass measurement. From the ¹³C-NMR (¹H decoupled and DEPT), UV and IR spectroscopic data it was evident that two of the four elements of unsaturation implied by the molecular formula of 1 could be attributed to a carbon-carbon double bond [δ 92.1 (C-8a, s), 172.3 (C-8, s, attached to oxygen)], and an α,β-unsaturated ester carbonyl [δ 174.2 (C-1, s)]; the molecule is thus bicyclic. The ¹H- and ¹³C-NMR spectra (see Table [1]) further revealed the presence of a secondary methyl [δ 1.39 (H₃ - 9, d)], and three methylene groups, as well as three methines, two of them attached to oxygen. In all, these data accounted for C₁₀H₁₂O₄, and showed the two unaccounted for H-atoms to be present as parts of two hydroxy functions. From the ¹H-¹H COSY spectrum of 1 a continuous chain of coupling was observed from H₃-9 through to H₂-7, and in so doing delineated the H₃-9 through to H₂-7, via H-4a, part of the molecule. From the ¹H-¹³C HMBC spectrum of 1 diagnostic HMBC correlations between the resonance of the 8-OH proton and those of C-7, C-8, and C-8a, showed this hydroxy group to bond to C-8 which further bonded to C-7 and C-8a. A further HMBC correlation, this time between the resonance for H₃ - 9 and that for C-1, showed C-3 to bond to the oxygen of the ester and enabled the ten-membered ring to be completed. As the remaining OH had to reside at C-5 (δ 66.0 d), C-4a had to bond to C-8a. With the planar structure of 1 established its relative configuration required resolution. As J _{H-3, H-4ax} = 11.3 Hz, it was evident that H-3 must have an axial orientation, similarly the magnitude of J _{H-4a, H-4ax}, 11.6 Hz, showed H-4a also to have an axial orientation, and gave the A ring a twist boat conformation. Finally, J _{H-4a, H-5} being less than 3 Hz revealed H-5 to have an equatorial orientation, and 1 to have the relative configuration as shown in 1. As 1 was crystalline it was possible to confirm the above structural deductions by single crystal x-ray analysis (see Fig. [1]). Attempts to make a crystalline heavy atom derivative of 1 so as to enable its absolute configuration to be established were unsuccessful. 5-Hydroxyramulosin (1) is a new natural product similar in structure to the aromatic compound 5-hydroxymellein, which is a common fungal secondary metabolite found in the phytopathogenic fungus Septoria nodorum Berk [12] as well as in the apple pathogen Botryosphaeria obtusa [13].

Together with 1, 7-methoxycoumarin (herniarin) was also isolated and identified by comparison of its ¹H- and ¹³C-NMR data with published values [11], [14]. Herniarin reduced the enzyme activity of HIV-1 RT to 86 % at a concentration of 66 μg/ml.[]

Materials and Methods

General experimental procedures were as previously published [2], [8]. Algal material was collected by hand using snorkel diving around the Azorean island of Faial, Atlantic Ocean, Portugal, March-April, 1997. The inner tissue of an algal sample was cut into small pieces and placed on agar plates containing isolation medium [15 g/L agar and 1000 mL sea water from the sample collecting site, and the antibiotics benzylpenicillin and streptomycin sulphate (250 μg/L)]. Fungal colonies growing out of the alga were transferred to medium for sporulation (1.0 g glucose, 0.1 g yeast extract, 0.5 g peptone from meat, enzymatic digest, 15 g agar, and 1000 mL sea water, pH 8) in order to enable taxonomic identification of the isolates. The fungal strain, Phoma tropica voucher number AZ14-4, was identified by Dr. S. Draeger, Institute for Microbiology, Technical University of Braunschweig.

P. tropica was cultured at 20 °C for 4 weeks in 14 L of liquid malt extract soy meal agar (30 g malt extract [Merck], 3 g peptone from soy meal, papain-digest [Merck], 1 L artificial sea water).

Inhibition of the enzyme HIV-1 reverse transcriptase was examined according to the methods of Kirsch et al. [9]. The positive control, foscarnet (10 μM), reduced enzyme activity to 17 %.

Prior to extraction with EtOAc (24 L) the fungal culture was blended using the Ultra Turrax T 25 at 8000 min^-1. The resultant EtOAc extract (3.9 g) was purified using normal phase silica gel (200 g, vacuum liquid chromatography [VLC]) employing step gradient elution from petroleum ether (PE) to EtOAc (from 100 % PE to 100 % EtOAc in 10 steps, each step 350 mL) to yield 15 fractions each of 250 mL. VLC fractions 7 - 12, in which antimicrobial activity was concentrated, were combined and separated again by VLC with 50 % PE/EtOAc to yield a further 14 fractions each of 100 mL. Fractions 7 and 8 of the second VLC were combined and separated by HPLC [Merck LiChromspher Si60, 5 μm, 250 × 7 mm eluted with PE : (Me)₂CO, 4 : 1], and further purified by RP-18 HPLC to yield 1 (26 mg, Knauer Spherisorb 5 ODS2, 5 μm, 250 × 8 mm eluted with MeOH : H₂O, 3 : 2). Fractions 5 and 6 of the second VLC were combined because of similar ¹H-NMR information and separated by normal phase HPLC to give 2 (20 mg, eluted with PE : (Me)₂CO, 4 : 1).

Single crystal X-ray crystallographic data for 5-hydroxyramulosin (1): Crystals of 1 suitable for single crystal X-ray diffraction analysis were obtained by recrystallisation from a 1 : 1 mixture of petroleum ether and (Me)₂CO. C₁₀H₁₄O₄; M_r = 198.0888, orthorhombic, P2₁2₁2₁, a = 4.7120(3) Å, b = 13.9376(10) Å, c = 14.2786(10) Å, Z = 4, D _x = 1.404 mg/m³, μ (Mo Kα) = 0.11 mm^-1, T = 143 K. A colourless plate of 1, approximately 0.5 × 0.25 × 0.1 mm, was mounted on a glass fibre and transferred to the cold gas stream of the diffractometer (Bruker SMART 1000 CCD). A total of 7010 intensities were recorded to 2Θ 57.5 °, of which 1393 were unique (Friedel opposites were merged). The structure of 1 (see Fig. [1]) was refined anisotropically on F₂ using the program SHELXL-97 [10]. The final wR₂ was 0.088, with R _int = 0.064 and R ₁ = 0.037, for 136 parameters; S = 1.01, max. Δρ = 0.26 eų. Hydroxy hydrogens were refined freely, methyls as rigid groups, and other hydrogens using a riding model. Within the crystal there is an intramolecular hydrogen bond from the hydroxy group O-8 to O-1. The latter atom also accepts an intermolecular H bond from the other hydroxy group at O-5 (via a 2₁ screw operator), leading to chains of molecules parallel to the z axis. Atomic co-ordinates, bond lengths and angles, and thermal parameters have been deposited at the Cambridge Crystallographic Data Center (University Chemical Laboratory, 12 Union Road, Cambridge CB2 1EZ, UK).

5-Hydroxyramulosin (1): White crystals; (1.9 mg/L); m. p. 191 °C; [α]_D ²⁰: + 17.1 ° (c 0.20, EtOH); UV (EtOH) λ_max (log ε) = 264 nm (3.62); IR (film): ν_max = 3450, 2980, 2910, 1625, 1590 cm^-1; EIMS m/z (rel. int.) = 198 [M⁺] (40), 180 (26), 154 (100), 136 (92), 121 (50), 111 (19), 95 (19), 85 (16), 68 (49), 55 (33), 43 (32); HREIMS m/z = 198.0887 (calcd. for C₁₀H₁₄O₄ 198.0888); ¹H- and ¹³C-NMR data see Table [1].

7-Methoxycoumarin (herniarin) (2): Spectroscopic data as previously published [11].

Acknowledgements

We thank Dr. S. Draeger, Institute for Microbiology, TU Braunschweig for taxonomic identification of the investigated fungal strain. We also thank Dr. V. Wray and his group, GBF Braunschweig, for recording all NMR spectra. Financial support from the Bundesministerium für Bildung und Forschung (BMBF), and Bayer AG, Leverkusen, is gratefully acknowledged.

References

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• 11 Zubía E, Luis F R, Massanet G M, Collado I G. An efficient synthesis of furanocoumarins.  Tetrahedron. 1992;  20 4239-46
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• 14 Osborne A G. ¹³C-NMR spectral studies of some methoxycoumarin derivatives. A re-assignment of citropten (limettin) and an examination of peri-proximity effects for the methyl-methoxy and methoxy-methyl couples.  Magnetic Resonance in Chemistry. 1989;  27 348-54
  CrossrefPubMedSearch in Google Scholar

Dr. Anthony D. Wright

Institute for Pharmaceutical Biology

University of Bonn

Nußallee 6

53115 Bonn

Germany

References

• 1 Höller U, König G M, Wright A D. A new tyrosine kinase inhibitor from a marine isolate of Ulocladium botrytis and new metabolites from the marine fungus Asteromyces cruciatus and Varicosporina ramulosa . European Journal of Organic Chemistry 1999: 2949-55
  Search in Google Scholar
• 2 Wegner C, Kaminsky R, König G M, Wright A D. Ascosalipyrrolidinone A, an antimicrobial alkaloid, from the obligate marine fungus Ascochyta salicorniae .  Journal of Organic Chemistry. 2000;  65 6412-17
  CrossrefPubMedSearch in Google Scholar
• 3 König G M, Wright A D. Marine natural products research: Current directions and future potential.  Planta Medica. 1996;  62 193-211
  PubMedSearch in Google Scholar
• 4 Soledade M, Pedras C, Taylor J L, Nakashima T T. A novel chemical signal from the ”Blackleg” fungus: Beyond phytotoxins and phytoalexins.  Journal of Organic Chemistry. 1993;  58 4778-80
  CrossrefPubMedSearch in Google Scholar
• 5 Capasso R, Evidente A, Vurro M. Cytochalasins from Phoma exigua var. heteromorpha .  Phytochemistry. 1991;  12 3945-50
  PubMedSearch in Google Scholar
• 6 Ichihara A, Oikawa H, Hashimoto M, Sakamura S, Haraguchi T, Nagano H. A phytotoxin, betaenone C, and its related metabolites of Phoma betae Fr.  Agricultural and Biological Chemistry. 1983;  47 2965-7
  PubMedSearch in Google Scholar
• 7 Wegner C, Schwibbe M, König G M, Wright A D. Differences between marine and terrestrial Phoma species as determined by HPLC-DAD and HPLC-MS.  Phytochemical Analysis.. 2000;  11 288-94
  CrossrefPubMedSearch in Google Scholar
• 8 Wright A D, König G M, Angerhofer C K, Greenidge P, Linden A, Desqueyroux-Faundéz R. Antimalarial activity: The search for marine derived natural products which demonstrate selective antimalarial activity.  Journal of Natural Products. 1996;  59 710-6
  CrossrefPubMedSearch in Google Scholar
• 9 Kirsch G, König G M, Wright A D, Kaminsky R. A new bioactive sesterterpene and antiplasmodial alkaloids from the marine sponge Hyrtios cf. erecta .  Journal of Natural Products. 2000;  63 825-9
  CrossrefPubMedSearch in Google Scholar
• 10 Sheldrick G M. 1997. SHELXL-97. University of Göttingen Göttingen, Germany;
  Search in Google Scholar
• 11 Zubía E, Luis F R, Massanet G M, Collado I G. An efficient synthesis of furanocoumarins.  Tetrahedron. 1992;  20 4239-46
  PubMedSearch in Google Scholar
• 12 Devys M, Barbier M, Bousquet J -F, Kollmann A. Isolation of the (-)-(3R)-5-hydroxymellein from the fungus Septoria nodorum .  Phytochemistry. 1994;  3 825-6
  PubMedSearch in Google Scholar
• 13 Venkatasubbaiah P, Chilton W S. Phytotoxins of Botryosphaeria obtusa .  Journal of Natural Products. 1990;  6 1628-30
  PubMedSearch in Google Scholar
• 14 Osborne A G. ¹³C-NMR spectral studies of some methoxycoumarin derivatives. A re-assignment of citropten (limettin) and an examination of peri-proximity effects for the methyl-methoxy and methoxy-methyl couples.  Magnetic Resonance in Chemistry. 1989;  27 348-54
  CrossrefPubMedSearch in Google Scholar

Dr. Anthony D. Wright

Institute for Pharmaceutical Biology

University of Bonn

Nußallee 6

53115 Bonn

Germany