Antifungal Polyketides and Other Compounds from Amazonian Endophytic Talaromyces Fungi

The continuous search for biologically active compound candidates pushes the pursuit of new substances produced by diverse organisms. Endophytic fungi are known as a promising source of metabolites with several biological activities. Based on the rich Amazonian biodiversity and the chemical potential of microbial sources, three Talaromyces strains, all major endophytes from their respective host plants, were studied aiming at the isolation of biologically active secondary metabolites. Through classical chromatographic approaches, 13 compounds were isolated from the antimicrobial extracts of the studied strains. From these, polyketides, steroids, anhydrides, and phenolic compounds were identified. Among them, two previously undescribed compounds were elucidated based on spectroscopic and spectrometric methods, a homodimer chromanone and a maleic anhydride methyl ester. The antimicrobial activity against a panel of pathogenic microorganisms from extracts, and isolated compounds was evaluated.


Introduction
The Amazon rainforest possesses the richest biodiversity of all the earth's ecosystems, which includes an enormous diversity of microorganisms. 1 From these, a vast range of fungi species play a vital role in the nutrient re-cycling, a vital process for the maintenance of the rainforest. 2 This is one of the keys to sustain the Amazonian ecosystem. 2 Among distinct types of fungi present in this environment, endophytes are receiving attention due to their ability to produce biologically active natural products. 3,4 Endophytic strains of Talaromyces are still poorly studied, with few works reporting mainly secondary metabolites from the alkaloid, 5 terpene, 6 and polyketide classes. 7 Some of the described compounds possess remarkable pharmacological activities. As examples, thermolides A-F from T. thermophilus with nematocidal activities comparable with commercial avermerctins 8 and the steroid derivative wortmannin from T. wortmannin, 9 which its semisynthetic derivatives are under phase II clinical trials for a new anticancer drug. 10 Our research group has been interested in the chemistry of Amazonian endophytic fungi due to their abilities to produce metabolites with great interest in their ecological associations. 11 In our continuing search for new substances, this study describes the chemical investigation of extracts produced by three endophytic strains belonging to the genus Talaromyces isolated from the roots of Duguetia stelechantha (strain DgCr2 2.1b), root barks of Rollinia mucosa (strain AnspCr1 1.1), and the trabecular structure of Victoria amazonica (strain VrTrb2 1.1). Vol. 29, No. 3, 2018 Experimental General One-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy data were acquired using an AVANCE 400 spectrometer (Bruker) operating at 9.4 T (400 and 100 MHz for 1 H and 13 C, respectively) and with an ASCEND 500 spectrometer (Bruker) at 11.7 T (500 and 125 MHz for 1 H and 13 C, respectively). Deuterated chloroform (CDCl 3 ), dimethyl sulfoxide (CD 3 SOCD 3 ), and methanol (CD 3 OD) (Cambridge Isotope Labs) were used as NMR solvents. The semi-preparative high-performance liquid chromatography (HPLC) was carried out on a Shimadzu UFLC system (LC-6 AD pump, DGU-20A5 degasser, SPD-20AV UV detector, rheodyne injector, CBM-20A communication module). Analytical and preparative thin-layer chromatography (TLC) were run on 0.25 mm thick aluminum-backed silica-gel 60 plates type F-UV254/365 (Merck). The spots were visualized by exposure to ultraviolet light at 254 or 366 nm, as well as by spraying with sulfuric vanillin reagent. High resolution electrospray ionization mass spectrometry (HRESIMS) measurements were recorded on a Waters Synapt HDMS instrument with a quadrupole timeof-flight (QTOF) geometry with an electrospray ionization (ESI) source. Optical rotations were obtained on a Jasco P-1020 polarimeter. All solvents used for chromatography and MS experiments were HPLC grade purchased from Tedia and water was purified by a Milli-Q system (Millipore).

Fungal material
The plants D. stelechantha and R. mucosa (Annonaceae) were collected at the experimental farm of the Universidade Federal do Amazonas, Manaus City, Amazonas State, Brazil. The plant material of Victoria amazonica (Nymphaeaceae) was collected at the km 20 of the BR-194 highway, at the city of Careiro da Várzea, Amazonas State, Brazil. The procedures for fungi isolation and purification followed previously determined protocols. 12 All isolated strains were identified according to traditional morphological criteria and by sequencing of the fungus internal transcribed spacer (ITS-1 to ITS-2) ribosomal deoxyribonucleic acid (rDNA). The obtained sequences were compared with previously deposited sequences from the GenBank. 13 Vouchers were deposited at the fungal collection of the LabMicra group of the Universidade Federal do Amazonas. Strain DgCr2 2.1b was identified as Talaromyces stipitatus, while strains AnspCr1 1.1 and VrTrb2 1.1 were designated as Talaromyces sp., possibly undescribed species.

Extract production
All the studied strains were inoculated into 60 Erlenmeyer flasks containing 300 mL of potato dextrose broth medium (PDL) (strains AnspCr1 1.1, and VrTrb2 1.1) or international Streptomyces project 2 liquid medium (ISP2) (strain DgCr2 2.1b). The incubation period was 28 days for T. stipitatus DgCr2 2.1b, 23 days for strain AnspCr1 1.1, and 30 days for strain VrTrb2 1.1, all growing at 28 °C. After the complete growth, the mycelial mass was separated from the fermented broth by a vacuum filtration procedure. For all fungi, the liquid portion was extracted with ethyl acetate followed by an ethyl acetate/isopropanol 8:2 (v:v) mixture. Fungal cells were extracted with an ethyl acetate/methanol 1:1 (v:v) mixture followed by methanol. For the extraction procedures, solvents were evaporated under reduced pressure affording the crude extracts.

Antimicrobial assay
The preliminary antibacterial activity of the obtained extracts was determined according to the agar-diffusion method with its methodology being previously described elsewhere. 11,15 The evaluation of the antimicrobial potential of pure compounds was assessed by the microbroth dilution method as recommended by the US National Committee for Clinical Laboratory Standards (NCCLS). 11 The inhibition halo (mm) for extracts and the minimal inhibitory concentrations (MICs, μg mL -1 ) for pure compounds were evaluated against a panel of pathogenic microorganisms. Wild hospital strains of gram-positive Bacillus cereus, Staphylococcus aureus, gram-negative Escherichia coli, and a wild environmental strain of the plant pathogen R. solanacearum were used for the antibacterial assays.
The antifungal potential was evaluated against a wild soil strain of Penicillium avellaneum, and wild hospital strains of Candida albicans and Candida tropicalis. Tetracycline and ampicillin (3 μg mL -1 ), and ketoconazole (4 μg mL -1 ) ( Table 3) were used as antibacterial and antifungal positive controls, respectively. Dimethyl sulfoxide (DMSO) 10% was used as negative control.

Artifact formation investigation
To evaluate the possibility of forming methyl esters of cordyanhydrides as artifacts during fractionation procedures with silica gel, we performed a test experiment. The strain Talaromyces sp. VrTrb2 1.1, was re-cultivated and extracted using the same methodology (ethyl acetate) in a smaller scale. The obtained extract was dried, re-suspended in isopropanol/water (8:2, v:v), and analyzed by direct infusion atmospheric pressure chemical ionization mass spectrometry (DI-APCI-MS) in the positive mode using an LCQ-fleet ion trap mass spectrometer (Thermo
In a previous investigation, Cai et al. 26 reported some xanthone dimers from the solid fermentation of a soil Alternaria strain, including chromanone homodimers with 6-6' linkage. However, authors reported that these derivatives were prone to degradation, thus, the stereochemistry investigations were discontinued. Moreover, these reported compounds named blennolides H and I displayed different coupling constant between H-9 and H-10 ( 3 J H-5, H-6 = 4.0 Hz) indicating different diastereomer analogues. 25 It is important to notice that the trivial names blennolides H and I were also used in other works, 16,25 but representing different structures (Figure 2c). Thus, compound 1 was assigned as a new chromanone homodimer, and to avoid any misunderstanding was named as being paecilin D, in respect to the previously isolated dimeric xanthones bearing two chromanone monomers. 19,27 Compound 7, obtained as a white amorphous solid from the strain VrTrb2 1.1 also displayed minor impurities, that could interfere in the optical activity. Its HRESIMS spectrum The coupling constants observed for these two signals indicated a double bond in trans orientation. 28 The presence of a heptet-like signal at d 2.21 (hept, J 6.8 Hz, H-7) was an indicative of a methinic hydrogen in a 3-ethylpentane system. Four double doublets at d 2.45 (dd, J 6.8, 13.9 Hz, H-6a), 2.59 (dd, J 6.8, 13.9 Hz, H-6β), 2.37 (dd, J 6.8, 13.9 Hz, H-8a), and 2.56 (dd, J 6.8, 13.9 Hz, H-8β) constituted two ABC-like spin systems. Beyond this, the presence of a typical methoxyl resonance (d 3.67, s) and two methyl groups (d 0.99 and 1.12) was observed ( Table 2).
To assess if 7 was an artifact, DI-APCI-MS fingerprinting was employed. The corresponding mass spectrum of the main metabolites produced by Talaromyces sp. VrTrb2 1.1 (in the Supplementary Information (SI) section) displayed the protonated molecule ions of the acidic version (cordyanhydride A, m/z 391) and its corresponding methyl ester (m/z 405), isolated for the first time in this communication. It is worth noticing that prior to the MS analysis methanol and silica gel were not used, reinforcing that compound 7 is not an artifact. Therefore, we concluded that 7 is a new naturally occurring compound.
Paecilins (1) are rare 2,2-disubstituted chroman-4-one compounds comprising monomers and biaryl single bonded dimers. Only three structures were previously described: paecilin A (homodimer) and paecilin B (monomer) isolated from Paecylomyces sp. (endophytic), 19 paecilin B in Setophoma terrestris (soil), 16 and paecilin C (heterodimer) in Penicillium sp. (marine organism). 27 This is the first report of all identified polyketides in endophytic Talaromyces strains.  Cordyanhydrides (7 and 8) were originally described from the insect pathogen fungus Cordyceps pseudomilitaris, 24 and more recently from Paecilomyces tenuipes, 29 and Dwayaangam colodena. 30 So far, compounds bearing maleic anhydride moieties such as telfairic and itaconitin anhydrides, in addition to regular cordyanhydrides are known as insecticidal compounds. 24 To the best of our knowledge a natural methyl ester from any of these compounds was not previously described.

Antimicrobial assays
Compounds 1-9 were tested for their antimicrobial activity against a panel of human and plant pathogens ( Table 3). All fractionated extracts displayed at least a moderate antimicrobial activity to be selected and further investigated. T. stipitatus DgCr2 2.1b showed to be a prolific source of polyketides, especially xanthone dimers. Bioactivity-guided fractionation of this sample displayed that sub-fraction sP-6 had antifungal potential against C. albicans and C. tropicalis (MIC, 15.6 μg mL -1 ). From this sample, five different polyketides were assayed for their antimicrobial activities, in which, only paecilin D (1) and versixanthone (4) were active against the yeasts (MIC, 15.6 and 31.3 μg mL -1 , respectively). The compound  blennolide G (3) displayed a weak antifungal activity against the same fungi (MIC, 125 μg mL -1 ). Paecilin B, the monomeric unit of compound 1 isolated from both Talaromyces sp. AnspCr1 1.1 and DgCr2 2.1b, showed a moderate antifungal activity (MIC, 62.6 μg mL -1 ), which indicates that dimerization enhances the activity. Other compounds, including the isolated cordyanhydrides were not active against the assayed pathogens (Table 3). Secalonic acids and its related compounds, paecillins and blennolides, are poorly studied with respect to their pharmacological properties, although their structures and biosynthesis are well known. 31 Anticancer activities were recorded for some derivatives against several tumor cell lines, where secalonic acid D is the most active. 32 Concerning antimicrobial activities, blennolides 21 and dicerandrols 33 inhibited several pathogens. So far, only paecilin C was evaluated for its antibacterial potential, however, showed no activity. 27 Therefore, this is the first report of the antifungal activity of a paecilin derivative.

Conclusions
The chemical investigation of the three Amazonian endophytic strains of Talaromyces afforded 13 different compounds. Among these, two undescribed compounds were fully characterized, the polyketide paecilin D (1) and the maleic anhydride cordyanhydride A methyl ester (7). The antimicrobial screening indicated that 1 and 4 (versixanthone) are promising antifungal agents. The obtained results highlight the immense potential of the Amazon rainforest to yield novel natural products as well as bioactive compounds.

Supplementary Information
Supplementary information is available free of charge at http://jbcs.sbq.org.br.