Scopularides Revisited: Molecular Networking Guided Exploration of Lipodepsipeptides in Australian Marine Fish Gastrointestinal Tract-Derived Fungi

Chemical analysis of a cultivation of an Australian Mugil mullet gastrointestinal tract (GIT) derived fungus, Scopulariopsis sp. CMB-F458, yielded the known lipodepsipeptides scopularides A (1) and B (2). A comparative global natural product social (GNPS) molecular networking analysis of ×63 co-isolated fungi, detected two additional fungi producing new scopularides, with Beauveria sp. CMB-F585 yielding scopularides C–G (3–7) and Scopulariopsis sp. CMB-F115 yielding scopularide H (8). Structures inclusive of absolute configurations were assigned by detailed spectroscopic and C3 Marfey’s analysis, together with X-ray analyses of 3 and 8, and biosynthetic considerations. Scopularides A–H (1–8) did not exhibit significant growth inhibitory activity against a selection of Gram positive (+ve) and negative (−ve) bacteria, a fungus, or a panel of three human carcinoma cell lines.


Introduction
As part of our ongoing investigation into secondary metabolites from Australian marine-derived fungi, we speculated that selected bottom feeding fish species may act as natural myco-accumulators, and as such could be a readily accessible source of marine-derived fungi. Using three Mugil mullet acquired from a local fish market, we assembled a library of~500 chemically-distinct gastrointestinal tract (GIT)-derived fungi. In a preliminary validation of the potential of this resource, we reported on an unprecedented class of hydrazine containing furano Schiff bases, the prolinimines, from Trichoderma sp. CMB-F563 [1]. Building on this achievement, we now describe an investigation into scopularide lipodepsipeptides, literature accounts of which are limited to scopularides A-B (1-2) from the marine sponge-derived fungus Scopulariopsis brevicaulis NCPF-2177 [2]. This current study was prompted by a fortuitous re-isolation of scopularides A-B (1-2) from the Mugil mullet GIT-derived Scopulariopsis sp. CMB-F458. Using 1 and 2 as authentic standards, a comparative global natural product social (GNPS) molecular networking [3] analysis of extracts obtained from ×63 co-isolated GIT-derived fungi, enabled the detection, and subsequent scaled-up cultivation, isolation and identification of the new scopularides C-G (3-7) from Beauveria sp. CMB-F585, and the new scopularide H (8) from Scopulariopsis sp. CMB-F115. Structures were assigned to 3-8 ( Figure 1) on the basis of detailed spectroscopic and chemical analysis, as summarised below.

Results and Discussion
The EtOAc extract from a M1S solid phase (20 × agar plate) cultivation of the Mugil mullet GIT-derived fungus Scopulariopsis sp. CMB-F458 was subjected to sequential solvent partitioning followed by reversed-phase HPLC to yield scopularides A-B (1-2). Planar structures for 1-2 were independently assigned by detailed spectroscopic analysis, with amino acid residues inclusive of absolute configurations assigned by C 3 Marfey's analysis ( Figures S46 and S47) [4]. The complete structures for 1-2 were confirmed by 1D NMR (methanol-d 4 ) in comparison to literature data (Tables S1 and S3) [2]. Armed with authentic standards of 1 and 2, a comparative GNPS analysis of extracts obtained from cultivations of ×63 co-isolated fungi detected a "scopularide" cluster incorporating nodes from Scopulariopsis sp. CMB-F458 and two other fungal strains, Beauveria sp. CMB-F585 and Scopulariopsis sp. CMB-F115 ( Figure 2).
HPLCs were performed using Agilent 1100 series HPLC instruments with corresponding detectors, fraction collectors and software inclusively. UPLC chromatograms were obtained on Agilent 1290 infinity UPLC system equipped with diode array multiple wavelength detector (Zorbax C8 RRHD 1.8 µm column, 50 × 2.1 mm, eluting with 0.417 mL/min 90% H 2 O/MeCN to 100% MeCN (with isocratic 0.01% TFA modifier) over 2.50 min). Ultra-high-performance-liquid-chromatography quadrupole-time-of-flight-mass-spectrometry (UHPLC-QTOF) analysis was performed on a UHPLC-QTOF instrument comprising an Agilent 1290 Infinity II UHPLC (Zorbax C 8 RRHD 1.8 µm column, 50 × 2.1 mm, eluting with 0.5 mL/min of isocratic 90% H 2 O/MeCN for 0.5 min followed by gradient elution to 100% MeCN over 4.5 min (with isocratic 0.1% formic acid modifier) coupled to an Agilent 6545 Q-TOF. MS/MS analysis was performed on the same instrument for ions detected in the full scan at an intensity above 1000 counts at 10 scans/s, with an isolation width of 4~m/z using a fixed collision energy and a maximum of 3 selected precursors per cycle.
Chiroptical measurements ([α]D) were obtained on a JASCO P-1010 polarimeter in a 100 × 2 mm cell at specified temperatures. Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker Avance 600 MHz spectrometer with either a 5 mm PASEL 1H/D-13C Z-Gradient probe or 5 mm CPTCI 1H/19F-13C/15N/DZ-Gradient cryoprobe, controlled by TopSpin 2.1 software. In all cases spectra were acquired at 25 • C in DMSO with referencing to residual 1 H or 13 C signals (δ H 2.50 and δ C 39.51 ppm) in the deuterated solvent, and in MeOH with referencing to residual 1 H or 13 C signals (δ H 3.31 and δ C 49.15 ppm) in the deuterated solvent. Electrospray ionization mass spectrometry (ESIMS) experiments were carried out on an Agilent 1100 series LC/MSD (quadrupole) instrument in both positive and negative modes. High-resolution ESIMS spectra were obtained on a Bruker micrOTOF mass spectrometer by direct injection in MeOH at 3 µL/min using sodium formate clusters as an internal calibrant.

Fungal Strain Isolation and Taxonomy
The fungal isolates CMB-F458, CMB-F585 and CMB-F115 were isolated from the gastrointestinal tract of a specimen of Mugil mullet fish, on M1 agar plates in presence of 3.3% artificial sea salt (M1S) incubated at 26.5 • C for 8 days. Genomic DNA for the three fungi were extracted from their corresponding mycelia using the DNeasy Plant Mini Kit (Qiagen) as per the manufacturers protocol. The 18s rRNA genes were amplified by PCR using the universal internal transcribed spacer primers ITS-1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS-4 (5 -TCCTCCGCTTATTGATATGC-3 ) purchased from Sigma-Aldrich. The PCR mixture (50 µL) contained 1 µL of genomic DNA (20-40 ng), 200 µM of each deoxynucleoside triphosphate (dNTP), 1.5 mM MgCl 2 , 0.3 µM of each primer, 1 U of Taq DNA polymerase (Fisher Biotec) and 5 µL of PCR buffer. PCR was performed using the following conditions: initial denaturation at 95 • C for 3 min, 30 cycles in series of 94 • C for 30 s (denaturation), 55 • C for 60 s (annealing) and 72 • C for 60 s (extension), followed by one cycle at 72 • C for 6 min. PCR products were purified with PCR purification kit (Qiagen, Victoria, Australia). Amplification products were examined by agarose gel electrophoresis. The DNA sequencing was performed by the Australian Genome Research Facility (AGRF) at the University of Queensland. BLAST analyses (NCBI database) on the ITS gene sequence for (a) CMB-F458 (GenBank accession no. MN080404) revealed 99% identity with the fungal strain Scopulariopsis sp.; (b) CMB-F585 (GenBank accession no. MN080403) revealed 98% identity with the fungal strain Beauveria sp.; and (c) CMB-F115 (GenBank accession no. MN080405) revealed 98% identity with the fungal strain Scopulariopsis sp.

C 3 Marfey's Analyses
Individual aliquots (50 µg) of 1-8 in 6 M HCl (100 µL) were heated to 100 • C in sealed vials for 8 to 12 h, after which the hydrolysates were concentrated to dryness at 40 • C under a stream of dry N 2 . The hydrolysates were then treated with 1 M NaHCO 3 (20 µL) and l-FDAA (1% solution in acetone, 40 µL) at 40 • C for 1 h, after which each reaction was neutralized with 1 M HCl (20 µL) and filtered (0.45 µm PTFE) prior to HPLC-DAD-ESIMS analysis. An aliquot (10 µL) of each analyte was injected into an Agilent Zorbax SB-C 3 column, 5 µm, 150 × 4.6 mm, 50 • C, with a 1 mL/min, 55 min linear gradient elution from 85% to 40% H 2 O/MeOH with a 5% isocratic modifier of 1% formic acid/MeCN. The analyte amino acid content was assessed by UV (340 nm) and ESI(±)MS monitoring, supported by SIE, with comparison to authentic standards. Amino acid standards were subjected to derivatization with l-and d-FDAA and HPLC-DAD-ESIMS analysis as described above for 1-8.

Global Natural Product Social (GNPS) Molecular Networking
A GNPS analysis of ×63 Mugil mullet GIT-derived fungal isolates was carried out on UPLC-QTOF-(+)MS/MS data acquired on EtOAc crude extracts. Aliquots (1 µL) of test solutions comprising 100 µg/mL of analyte in 100 µL MeOH were analysed on an Agilent 6545 Q-TOF LC/MS equipped with an Agilent 1290 infinity II UPLC system, utilising an Agilent SB-C 8 1.8 µm, 2.1 × 50 mm column, 0.5 min isocratic elution of 90% H 2 O/MeCN followed by 4.5 min gradient elution to 100% MeCN with a flow rate of 0.5 mL/min and a constant isocratic 0.1% formic acid/MeCN modifier. UPLC-QTOF-(+)MS/MS data acquired for all samples at collision energy of 40 eV were converted from Agilent MassHunter data files (.d) to mzXML file format using MSConvert software, and transferred to the GNPS server (gnps.ucsd.edu). Molecular networking was performed using the GNPS data analysis workflow using the spectral clustering algorithm with a cosine score of 0.7 and a minimum of 6 matched peaks. The resulting spectral network was imported into Cytoscape version 3.5.1, where nodes corresponding to media components and solvent were subtracted. Remaining nodes represented parent (+) m/z of metabolites detected in analysed extracts, with node size indicating metabolite abundance and edge thickness corresponding to cosine scores ( Figure 2, Figure 3, Figure 8, Figures  S53 and S54). Figure S53 represents the molecular network for ×63 GIT-derived fungal isolates, with the "scopularides" cluster represented in Figure 2. Similarly, scopularides molecular family clusters detected in CMB-F585, CMB-F115 and CMB-F458 crude extracts are represented in Figure 3, Figure 8 and Figure S54.
The data were deposited to the GNPS-MassIVE Datasets and are publicly accessible through the links listed below: Fish microbial library molecular networking MASSIVE: MSV000084190 (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=479a543e9b45408bbe93414769058784).