Teratopyrones A–C, Dimeric Naphtho-γ-Pyrones and Other Metabolites from Teratosphaeria sp. AK1128, a Fungal Endophyte of Equisetum arvense

Bioassay-guided fractionation of a cytotoxic extract derived from a solid potato dextrose agar (PDA) culture of Teratosphaeria sp. AK1128, a fungal endophyte of Equisetum arvense, afforded three new naphtho-γ-pyrone dimers, teratopyrones A–C (1–3), together with five known naphtho-γ-pyrones, aurasperone B (4), aurasperone C (5), aurasperone F (6), nigerasperone A (7), and fonsecin B (8), and two known diketopiperazines, asperazine (9) and isorugulosuvine (10). The structures of 1–3 were determined on the basis of their spectroscopic data. Cytotoxicity assay revealed that nigerasperone A (7) was moderately active against the cancer cell lines PC-3M (human metastatic prostate cancer), NCI-H460 (human non-small cell lung cancer), SF-268 (human CNS glioma), and MCF-7 (human breast cancer), with IC50s ranging from 2.37 to 4.12 μM while other metabolites exhibited no cytotoxic activity up to a concentration of 5.0 μM.


Introduction
Fungal endophytes constitute an abundant and underexplored group of fungi that inhabit plants in diverse natural and human-managed ecosystems [1,2]. These symbiotic fungi often produce bioactive metabolites, some of which may improve the growth or resiliency of the host plant [3]. Recent studies have demonstrated that fungal endophytes are rich sources of small-molecule natural products with novel structures and biomedical potential [4,5]. In our continuing search for bioactive and/or novel metabolites from endosymbiotic microorganisms [6], we have investigated a cytotoxic extract of the fungal endophyte, Teratosphaeria sp. AK1128, isolated from a photosynthetic stem of Equisetum arvense (field horsetail, Equisetaceae). Herein, we report cytotoxicity assay-guided fractionation of this extract, resulting in isolation and characterization of ten metabolites, including three new naphtho-γ-pyrone dimers, teratopyrones A-C (1-3), and the constituent responsible for cytotoxic activity, nigerasperone A (7). Teratosphaeria is a genus within the newly established fungal family Teratosphaeriaceae (Dothideomycetes, Ascomycota), which has been distinguished recently from Mycosphaerella (Mycosphaerellaceae) [7]. To the best of our knowledge, this constitutes the second report on metabolites of a fungal strain of the family Teratosphaeriaceae [8].
from Mycosphaerella (Mycosphaerellaceae) [7]. To the best of our knowledge, this constitutes the second report on metabolites of a fungal strain of the family Teratosphaeriaceae [8].

Results and Discussion
The EtOAc extract of a PDA (potato dextrose agar) culture of Teratosphaeria sp. AK1128 exhibiting cytotoxic activity, on bioassy-guided fractionation involving solvent-solvent partitioning, Sephadex LH-20 size-exclusion and silica gel chromatography followed by HPLC purification, afforded metabolites 1-10 ( Figure 1). Of these, 4-10 were previously known and were identified as naphtho-γ-pyrones, aurasperone B (4) [9], aurasperone C (5) [10], aurasperone F (6) [11], nigerasperone A (7) [12], and fonsecin B (8) [9], and diketopiperazines, asperazine (9) [13] and isorugulosuvine (10) [14], by comparison of their spectroscopic ( 1 H NMR, 13 C NMR, and LR-MS) data with those reported. Spectroscopic ( 1 H and 13 C NMR, HRESIMS, and UV) data of teratopyrones A-C (1-3), together with their common molecular formula, C31H26O11, suggested that they are dimeric naphtho-γ-pyrones [11]. In their 1 H and 13 C NMR spectra, two sets of signals were observed in different intensity ratios and this was suspected to be due to the atropisomerism around the C7-C10′ axis [10] and/or the presence of C-2 and C-2′ hemi-ketal stereoisomeric mixtures as a result of non-enzymatic formation of this moiety during the biosynthesis of naphtho-γ-pyrones [15]. Although several recent publications on dimeric naphtho-γ-pyrones report only one set of the NMR data, careful examination of the spectra of these dimeric naphtho-γ-pyrones provided in the Supporting Information of these papers indicated the presence of two sets of signals, corresponding to two possible tautomers [16][17][18]. The atropisomerization around the C7-C10′ axis is known to be restricted under mild conditions [10], and the atropisomer ratio for a given dimeric naphtho-γ-pyrone may depend on its producer fungus [19]. Although it is not possible to obtain the atropisomer ratio directly, the use circular dichroism (CD) data for the assignment of stereochemistry of the binaphthyl moiety of the major atropisomer of dimeric naphtho-γ-pyrones by the exciton chirality method has been reported [20,21]. Spectroscopic ( 1 H and 13 C NMR, HRESIMS, and UV) data of teratopyrones A-C (1-3), together with their common molecular formula, C 31 H 26 O 11 , suggested that they are dimeric naphtho-γ-pyrones [11]. In their 1 H and 13 C NMR spectra, two sets of signals were observed in different intensity ratios and this was suspected to be due to the atropisomerism around the C 7 -C 10 axis [10] and/or the presence of C-2 and C-2 hemi-ketal stereoisomeric mixtures as a result of non-enzymatic formation of this moiety during the biosynthesis of naphtho-γ-pyrones [15]. Although several recent publications on dimeric naphtho-γ-pyrones report only one set of the NMR data, careful examination of the spectra of these dimeric naphtho-γ-pyrones provided in the Supporting Information of these papers indicated the presence of two sets of signals, corresponding to two possible tautomers [16][17][18]. The atropisomerization around the C 7 -C 10 axis is known to be restricted under mild conditions [10], and the atropisomer ratio for a given dimeric naphtho-γ-pyrone may depend on its producer fungus [19]. Although it is not possible to obtain the atropisomer ratio directly, the use circular dichroism (CD) data for the assignment of stereochemistry of the binaphthyl moiety of the major atropisomer of dimeric naphtho-γ-pyrones by the exciton chirality method has been reported [20,21].

Fungal Isolation and Identification
In June 2008, a healthy individual of Equisetum arvense was collected from Beringian tundra in the Seward Peninsula of Western Alaska (64 • 30 04" N, 165 • 24 23" W; 6 m.a.s.l.) [23]. The photosynthetic Molecules 2020, 25, 5058 6 of 10 stem was washed in tap water and cut into ca. 2 mm 2 segments that were surface-sterilized by agitating sequentially in 95% EtOH for 30 s, 0.5% NaOCl for 2 min, and 70% EtOH for 2 min [23]. Forty-eight tissue segments were surface-dried under sterile conditions and then placed individually onto 2% malt extract agar (MEA) in sterile 1.5 mL micro centrifuge tubes. Tubes were sealed with Parafilm TM and incubated under ambient light/dark conditions at room temperature (ca. 21.5 • C) for up to one year. Emergent fungi were isolated into pure culture on 2% MEA, vouchered in sterile water, and deposited as living vouchers at the Robert L. Gilbertson Mycological Herbarium at the University of Arizona. One fungus of interest, isolate AK1128, was used for the present study. This fungus has been deposited at the University of Arizona Robert L. Gilbertson Mycological Herbarium (accession number AK1128).
Total genomic DNA was isolated from fresh mycelium of the isolate AK1128 and the nuclear ribosomal internal transcribed spacers and 5.8s gene (ITS rDNA; ca. 600 base pairs (bp)) and an adjacent portion of the nuclear ribosomal large subunit (LSU rDNA; ca. 500 bp) was amplified as a single fragment by PCR. Positive amplicons were sequenced bidirectionally as described previously [23]. A consensus sequence was assembled and basecalls were made by phred [24] and phrap [25] with orchestration by Mesquite [26], followed by manual editing in Sequencher (Gene Codes Corp.). The resulting sequence was deposited in GenBank (accession JQ759476).
Because the isolate did not produce diagnostic fruiting structures in culture, two methods were used to tentatively identify isolate AK1128 via molecular sequence data. First, the LSU rDNA portion of the sequence was evaluated using the naïve Bayesian classifier for fungi [27] available through the Ribosomal Database Project (http://rdp.cme.msu.edu/). The Bayesian classifier estimated placement within the Capnodiales (Dothideomycetes) with high support, but placement at finer taxonomic levels was not possible. Therefore, the entire sequence was compared against the GenBank database using BLAST [28]. The top ten BLAST matches were to unidentified dothideomycetous endophytes or uncultured ascomycetous clones, except for two strains of Colletogloeopsis dimorpha (strains CBS 120085 and CBS 120086). The matches to Colletogloeopsis dimorpha had 97% coverage and a maximum identity of only 92%, and similar levels of match precision to other taxa restricted our taxonomic inference. Therefore, to clarify the phylogenetic placement and taxonomic assignment of AK1128, two phylogenetic analyses were conducted. First, the top 99 BLAST matches were downloaded from GenBank, four problematic sequences for which quality was suspect were removed, and AK1128 and the resulting dataset was aligned automatically via MUSCLE (http://www.ebi.ac.uk/Tools/msa/ muscle/) with default parameters. The alignment consisted of dothideomycetous endophytes as well as described species of Dothideomycetes that mostly comprised taxa affiliated with some lineages recognized within Mycosphaerella (e.g., Teratosphaeria, Colletogloeopsis, Catulenostroma, etc.). The alignment was trimmed so that starting and ending points were generally consistent with the sequence length for AK1128 and adjusted manually in Mesquite [26] prior to analysis. The final dataset consisted of 96 sequences and 1084 characters. This dataset was analyzed by maximum likelihood in GARLI (Zwickl, D. J. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion (Ph.D. Dissertation, The University of Texas at Austin, Austin, TX, USA, 2008) using the GTR + I + G model of evolution as determined by ModelTest [29]. The resulting topology indicated that AK1128 had an affinity for Teratosphaeria, Catenulostroma, and relatives but was not phylogenetically affiliated with Colletogloeopsis (data not shown). Because this analysis included many unknown taxa (endophytes) and we could not root the tree with certainty, the taxon sampling was found to be insufficient to infer with confidence the placement of the AK1128.
A second phylogenetic analysis was therefore conducted using taxa affiliated with Teratosphaeria, Catulenostroma, and relatives, as analyzed previously [30]. The alignment of ITS rDNA sequences from [30] was obtained from TreeBase and pruned to include only those taxa of interest based on our analysis above (i.e., the lower half of Figure 1 in [30] in a preliminary analysis, and then only those taxa most closely related within Teratosphaeria in the final analysis). The sequence for AK1128 was integrated into the pruned dataset and the data were realigned, adjusted, and analyzed as described above. The resulting dataset consisted of 79 sequences and 582 characters in the preliminary analysis. A bootstrap analysis with 1000 replicates was conducted to assess topological support and the topology was rooted with Devriesia strelitziae [30]. The resulting tree suggested that AK1128 is affiliated with Teratosphaeria species (data not shown).
Therefore, the dataset was pruned further, focusing only on those Teratosphaeria species suggested to be close relatives of AK1128. The final analysis included 42 terminal taxa and 554 characters and was rooted with Catulenostroma macowanii based on the topology of the preliminary analysis. The final tree with maximum likelihood bootstrap values is shown in Supplemental Figure S13. The final analysis placed AK1128 with certainty within the genus Teratosphaeria (Teratosphaeriaceae), likely in affiliation with T. associata (known from Eucalyptus [31] and Protea [30] from Australia). Given the distinctive geographical origin and host of AK1128, and the sister relationship of AK1128 to known strains of T. associata (Supplemental Figure S13), we designate the strain as Teratosphaeria sp. AK1128, affiliated with but distinct from known variants of T. associata.

Conclusions
Ten metabolites, including three new dimeric naphtha-γ-pyrones, teratopyrones A-C (1-3), were isolated from a PDA culture of the endophytic fungus, Teratosphaeria sp. AK1128. This constitutes the second report of the occurrence of secondary metabolites in a fungus of the family Teratosphaeriaceae. The ECD spectra of teratopyrones A-C suggested that all three of them have negative exciton chirality. Cytotoxicity data for nigerasperone A (7) from this and previous studies suggest that it has selective activity for certain cancer cell lines.