Analogs of the carotane antibiotic fulvoferruginin from submerged cultures of a Thai Marasmius sp.

A recent find of a Marasmius species in Northern Thailand led to the isolation of five unprecedented derivatives of the carotane antibiotic fulvoferruginin (1), fulvoferruginins B–F (2–6). The structures of these sesquiterpenoids were elucidated using HRESIMS, 1D and 2D NMR, as well as CD spectroscopy. Assessing the bioactivity, fulvoferruginin emerged as a potent cytotoxic agent of potential pharmaceutical interest.


Introduction
The family Marasmiaceae (Agaricomycetes, Basidiomycota) presently contains ten genera with Marasmius Fr. being the predominant one (currently comprising over 600 recognized species). Most of these fungi are considered saprotrophs, even though some (e.g., Moniliophthora species) are plant pathogens [1]. They are rather ubiquitous, but have been frequently overlooked owing to the small dimensions of their basidiomes. As of now, their taxonomy is not well settled, and a world monograph using modern mycological methodology is still not available.
As of 2006, the family forms a sister taxon to the Omphalotaceae within the suborder of Marasmiineae (Agaricales) [2,3]. Among Basidiomycota, the Marasmiineae are most famous for their abundant chemical diversity. Metabolites with various bioactivities described from Marasmius spp. include the cryptoporic acids [4], marasmals, marasmones, and oreadones [5,6], the caryophyllane hebelophyllene C [7], and the carotane fulvoferruginin (1) [8] (Figure 1). The latter is the only known secondary metabolite from M. fulvoferrugineus Gilliam and displays a modest antibiotic and moderate antifungal activity. A recent find of a Marasmius species in Northern Thailand led, however, to the isolation and identification of five novel derivatives of this carotane-type sesquiterpenoid. The present study is dedicated to describing their isolation, and biological and physicochemical characterization.
The methyl group C-14 of fulvoferruginin B (2) results in an additional stereocenter at C-11. The proton at C-11 (δ H 2.72) exhibited 1 H, 1 H ROESY correlations with H-5 (δ H 4.95) and H 3 -13 (δ H 0.99) indicating that they are on the same face of the molecule. These ROESY correlations are otherwise identical to those observed for fulvoferruginin (1). The relative stereochemistry at C-6 and C-10 was further confirmed through comparison with ROESY correlations of metabolite 3 ( Figure 3). . It differed from compound 2 only in the presence of a carboxyl group (δ C 171.9) instead of the tertiary methyl C-14, and a methine (C-10, δ C 39.3) instead of a hydroxylated quaternary carbon. The presence of the methine C-11 was confirmed by measuring 1D/2D NMR spectra of 3 in chloroform-d (see Table  S1 in Supporting Information File 1). The corresponding proton H-11 (δ H 3.38) forms again a spin system with H-5 (δ H 4.89) and H-13 (δ H 1.01) in the COSY spectrum ( Figure 2). Furthermore, the relative configurations of C-6 and C-10 were elucidated by the ROESY correlation ( Figure 3) depicted between H-6 (δ H 2.57) and H-10 (δ H 2.75). All these correlations also support the structure of compound 2.   Table S1 in Supporting Information File 1).  Table 1 and Supporting Information File 1). Fulvoferruginin E (5), isolated as a clear solid, shares the same molecular formula C 15 H 22 O 4 as compound 2, as deduced from the HRESIMS spectrum also exhibiting a protonated molecular ion peak at m/z 267.1589 [M + H] + . Its 1D/2D NMR spectroscopic data are similar to those of metabolite 4, with the only striking variation being the replacement of two olefinic methines (C-1/C-2) with methylenes (C-1: δ C 41.0, C-2: δ C 31.7). The absence of this double bond has previously been observed in the structurally related hercynolactone from the liverworts Barbilophozia lycopodioides and B. hatcheri [9].
To add to this, another metabolite, fulvoferruginin F (6), with a molecular formula of C 15 H 20 O 3 was isolated that is also lacking the double bond at C-1/C-2. It further possesses an additional double bond between C-10/C-11, just like hercynolactone. However, NMR data analysis disclosed that the position C-9 (δ C 70.8) is hydroxylated. The ROESY spectrum indicates H-9 (δ H 4.88) to be cofacial with H-5 and H-13. Thus, fulvoferruginin F could also be named 9-hydroxyhercynolactone [9].
Assessing the bioactivity of the fulvoferruginins A-F (1-6), aside from the known antifungal activity of fulvoferruginin (1), no other antimicrobial activities were observed (Table S2 in Supporting Information File 1). All metabolites were also tested against the murine fibroblast cell line L929 and the cervix carcinoma cell line KB3.1. While all metabolites exhibited very weak cytotoxic effects at the highest concentration, only 2, 4, and 6 displayed mild cytotoxicity, allowing for a determination of IC 50 values, which ranged from 9.5-32 µg/mL. To our surprise, fulvoferruginin (1) displayed greater cytotoxic effects than previously reported (though for other cell lines) [8], which led us to assess its cytotoxicity further against different carcinoma cell lines. The IC 50 values of compound 1 range from 0.06-0.7 µg/mL for all tested cell lines (Table S2 in Supporting Information File 1).

Discussion
The lack of bioactivity for metabolites 2-6 can be attributed to an absence of the α-methylene lactone unit present in fulvoferruginin (1). Nevertheless, the cytotoxicity detected here for fulvoferruginin shows that re-evaluating the bioactivity of previously isolated basidiomycete metabolites in different bioassays can lead to unexpected results of potential pharmaceutical interest.
Klein et al. obtained a crystal structure of fulvoferruginin (1) [8], as did Huneck et al. for hercynolactone [9], verifying their relative configuration. Huo et al. have further confirmed the absolute configuration of compound 1 by utilizing the CD exciton chirality method. Additionally, our recorded CD spectra ( Figure S1 in Supporting Information File 1) of metabolites 3 and 4, are in close agreement with 1. As the metabolites 2-6 displayed analogous relative stereochemistry and optical rota-tions, we presume that the new compounds arise from the same biosynthetic genes as the parent compound, and postulate that the congeners should also have the same absolute configuration as compound 1 [10].
For a long time, fulvoferruginin was only known to be produced by a strain of M. fulvoferrugineus found in Northern America, but there is a recent report on the occurrence of fulvoferruginin (1) in a basidiomycete collected in China. This strain was tentatively identified as Gymnopus sp. through analysis of its internal transcribed spacer (ITS1-5.8s) rDNA region [10]. Today, Gymnopus belongs to the Omphalotaceae and some Marasmius species have been reassigned to Gymnopus, as have certain Gymnopus spp. to genera like Marasmiellus. However, intensive phylogenetic studies on these genera remain to be conducted. A macroscopic differentiation to Marasmius is difficult and a taxonomic classification based on ITS alone has proven to be insufficient for these genera [11]. In addition, the recent finding that multiple copies of the rDNA can be present in one and the same genome of certain fungi, leading to up to more than 10% deviations [12], makes us suspicious about the validity of the previous classification of the Chinese "Gymnopus" species.
Taking also other marker loci into consideration, as is required for publishing a new fungal species [12] The DNA extraction was performed using an EZ-10 Spin Column Genomic DNA Miniprep kit (Bio Basic Canada Inc., Markham, Ontario, Canada) following the manufacturer's protocol. A Precellys 24 homogenizer (Bertin Technologies, France) at 6000 rpm for 2 × 40 s was used for cell disruption. DNA regions were amplified using standard primers following our previously published protocols [13]. For both DNA regions, the PCR products were purified utilizing the Nucleo Spin ® Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). Sequencing of the PCR products was carried out at the Department of Genome Analytics of the Helmholtz Centre for Infection Research, Braunschweig, Germany.

Fermentation and isolation of metabolites 1-6
Cultures of Marasmius sp. strain MFLUCC 14-0681 were maintained on YMG agar, as described by Klein et al. [8]. A 100 mL seed culture of the strain in ZM½ medium [14] was used to inoculate a 5 L batch fermentation in 25 500 mL Erlenmeyer flasks with ZM½ media for 15 days at 140 rpm and 23 °C. Afterwards, the fermentation broth was filtered and the supernatant extracted with 2% Amberlite TM XAD 16N (Rohm & Haas, Frankfurt a. M., Germany), subsequently resuspended in acetone and evaporated to dryness, resulting in 2.5 g of crude extract. This supernatant crude extract was filtered using a SPME Strata TM -X 33 u Polymeric RP cartridge (Phenomenex, Inc., Aschaffenburg, Germany) and subsequently pre-fractionated utilizing RP-HPLC with a Gilson PLC 2250 purification system (Middleton, WI, USA) and a VP Nucleodur 100-5 C 18 ec 250 × 40 mm, 7 µm column (Macherey-Nagel, Düren, Germany). Acetonitrile + 0.1% formic acid and deionized water + 0.1% FA served as mobile phase, running a gradient of 10 min at 5% acetonitrile + 0.1% FA, then increasing to 100% within 70 min, flow rate: 40 mL/min, UV detection at 200-600 nm. Pre-fractions were further separated using a Luna C18(2) 250 × 21 mm, 7 μm column (Phenomenex, Aschaffenburg, Germany) and deionized water + 0.05% TFA (solvent A) and acetonitrile + 0.05% TFA (solvent B) as mobile phase and a flow rate of 18 mL/min. Gradients were established individually for each pre-fraction as follows: a pre-fraction at 36 to 37 minutes was further separated with a gradient of 20-50% solvent B within 60 minutes, resulting in a peak of metabolite 4 (32 mg) at 27-27.5 min. The pre-fraction at 38 minutes was separated with a gradient from 20-70% B within 60 minutes of which a peak at 24-25 min resulted in metabolite 5 (1.6 mg). A pre-fraction from 42-43 min was further separated with a gradient from 30-60% B within 60 min, resulting in a major peak at 26-27 min being fulvoferruginin B (2, 16 mg) as well as a minor peak at 27-28 min, being fulvoferruginin (1, 9.8 mg). Yet another pre-fraction at 45 min was further separated using a gradient of 30% solvent B for 10 minutes then increasing to 75% B within 45 min. A peak at 30-30.5 min resulted in fulvoferruginin C (3, 28 mg). The pre-fraction peak at 41-42 minutes was further separated with a gradient of 30% solvent B for 20 min, then increased from 30 to 50% B within 40 min using an XBridge TM Trifunctional C18, 250 × 19 mm, 135 Å, 5 μm column (Waters, Eschborn, Germany) as stationary phase, resulting in a peak of metabolite 6 (1.2 mg) at 21-21.5 minutes.  Table 1 and copies of spectra are provided in Supporting Information File 1.

Antimicrobial activity
The minimum inhibitory concentrations (MIC) of metabolites 1-6 were assessed using a serial dilution assay in 96-well microtiter plates with YM6.3 media (10 g/L malt extract, 4 g/L glucose, 4 g/L yeast extract, pH 6.3) for filamentous fungi and yeasts, and with BD Difco TM Mueller Hinton Broth for bacteria. The antimicrobial assays were performed as previously described [13].

Supporting Information
Supporting Information File 1 HRESIMS profiles and copies of NMR spectra for compounds 1-6 in CD 3 OD, and for metabolite 3 also in CDCl 3 ; minimum inhibitory concentrations (MIC) of 1-6 for bacteria, yeasts and fungi as well as half inhibitory concentrations (IC 50 ) for different cell lines.