Noonindoles A–F: Rare Indole Diterpene Amino Acid Conjugates from a Marine-Derived Fungus, Aspergillus noonimiae CMB-M0339

Analytical scale chemical/cultivation profiling prioritized the Australian marine-derived fungus Aspergillus noonimiae CMB-M0339. Subsequent investigation permitted isolation of noonindoles A–F (5–10) and detection of eight minor analogues (i–viii) as new examples of a rare class of indole diterpene (IDT) amino acid conjugate, indicative of an acyl amino acid transferase capable of incorporating a diverse range of amino acid residues. Structures for 5–10 were assigned by detailed spectroscopic and X-ray crystallographic analysis. The metabolites 5–14 exhibited no antibacterial properties against G-ve and G+ve bacteria or the fungus Candida albicans, with the exception of 5 which exhibited moderate antifungal activity.

HRESIMS analysis of 5 revealed a molecular formula (C 34 H 46 N 2 O 6 , ∆mmu +2.7) requiring thirteen double bond equivalents (DBE). The NMR (methanol-d 4 ) data for 5 (Tables 1 and S2, and Figures S10-S15) disclosed resonances for ten sp 2 olefinic carbons and two sp 2 ester/lactone carbonyls, accounting for seven DBE and requiring that 5 be hexacyclic, while diagnostic 2D NMR correlations ( Figure 5) established a carbon/hetero skeleton in common with 1 further annotated by an N,N-dimethyl-valinyloxy ester pendant to C-14. The structure and absolute configuration for noonindole A (5) were confirmed by a single crystal X-ray analysis ( Figure 6 and Table S13). Mar. Drugs 2022, 20, x FOR PEER REVIEW 3 of 16  HRESIMS analysis of 5 revealed a molecular formula (C34H46N2O6, Δmmu +2.7) requiring thirteen double bond equivalents (DBE). The NMR (methanol-d4) data for 5 (Tables 1 and S2, and Figures S10-S15) disclosed resonances for ten sp 2 olefinic carbons and two sp 2 ester/lactone carbonyls, accounting for seven DBE and requiring that 5 be hexacyclic, while diagnostic 2D NMR correlations ( Figure 5) established a carbon/hetero skeleton in common with 1 further annotated by an N,N-dimethyl-valinyloxy ester pendant to C-14. The structure and absolute configuration for noonindole A (5) were confirmed by a single crystal X-ray analysis ( Figure 6 and Table S13).
Co-isolation of noonindoles A-F (5-10) and the known IDTs 11-14 together with an X-ray crystal analysis of 5 supported a common absolute configuration across the hexacyclic indole terpene core, and established the configuration of the N,N-dimethyl-L-valinyloxy moiety in 5. Although low yields combined with N-alkylation precluded hydrolysis and independent assignment of the amino acid residue absolute configuration in 6-9 (i.e., Marfey's analysis), an amino acid L configuration across 6-9 was proposed based on the likelihood of a common NRPS-like aminoacyl modifying enzyme in the noonindole biosynthetic gene cluster (BGC) (see below).
GNPS analysis of the EtOAc extract of the analytical scale D400 (MATRIX) culture of CMB-M0339 detected 5-9 along with associated nodes for a selection of putative minor analogues (Figure 7). The MS/MS spectra for 5-9 ( Figures S72-S75) revealed three common fragmentations attributed to loss of water ( Figure 8A), retro-Aldol loss of acetone ( Figure 8B), and loss of the amino acid residue ( Figure 8C). While low yields precluded isolation of the minor analogues i-viii, diagnostic MS/MS fragmentations and highresolution mass measurements (i.e., molecular formulae) permitted tentative assignments for i-v (Figures S74, S76-S79, Table S12) and on the basis of GNPS co-clustering and biosynthetic considerations to vi-viii, albeit with some allowance isomeric alternatives (Tables 3 and S12). The diversity of IDT amino acid conjugates produced by CMB-M0339 is in stark contrast to existing knowledge, which is limited to 3 from Aspergillus nomius [9] and 4 from A. alliaceus [10]. Unlike these earlier published accounts, it appears CMB-M0339 employs an NRPS-like aminoacylation enzyme with an adenylation domain tolerant of different amino acid substrates (i.e., Val, Leu, Ile, Pro, Ser, Thr, and homo-Ala).
A preliminary assessment of the noonindole biosynthetic gene cluster (BGC) suggests a biogenetic relationship linking 5-14 and inclusive of the minor co-metabolites i-viii starting with emindole SB (14) undergoing stereospecific epoxidation and ring closure to paspaline (11) followed by sequential oxidation to paspaline B (12) and the carboxylic acid 13, followed by decarboxylation and oxidation to paxilline (1) (Figure 9). Oxidation of 1 could then yield noonindole F (10) with further oxidation and/or amino acid acylation returning noonindoles A-E (5-9) and co-clustering minor analogues (i-viii). Consistent with this hypothesis, close examination of the CMB-M0339 D400 extract GNPS and UPLC-DAD-MS data using single ion extraction (SIE) detected an ion with a molecular formula attributable to 1 ( Figure S71). biosynthetic considerations to vi-viii, albeit with some allowance isomeric alternatives (Tables 3 and S12). The diversity of IDT amino acid conjugates produced by CMB-M0339 is in stark contrast to existing knowledge, which is limited to 3 from Aspergillus nomius [9] and 4 from A. alliaceus [10]. Unlike these earlier published accounts, it appears CMB-M0339 employs an NRPS-like aminoacylation enzyme with an adenylation domain tolerant of different amino acid substrates (i.e., Val, Leu, Ile, Pro, Ser, Thr, and homo-Ala).  A preliminary assessment of the noonindole biosynthetic gene cluster (BGC) suggests a biogenetic relationship linking 5-14 and inclusive of the minor co-metabolites i-viii starting with emindole SB (14) undergoing stereospecific epoxidation and ring closure to paspaline (11) followed by sequential oxidation to paspaline B (12) and the carboxylic acid 13, followed by decarboxylation and oxidation to paxilline (1) (Figure 9). Oxidation of 1 could then yield noonindole F (10) with further oxidation and/or amino acid acylation returning noonindoles A-E (5-9) and co-clustering minor analogues (iviii). Consistent with this hypothesis, close examination of the CMB-M0339 D400 extract GNPS and UPLC-DAD-MS data using single ion extraction (SIE) detected an ion with a molecular formula attributable to 1 ( Figure S71). (Tables 3 and S12). The diversity of IDT amino acid conjugates produced by CMB-M0339 is in stark contrast to existing knowledge, which is limited to 3 from Aspergillus nomius [9] and 4 from A. alliaceus [10]. Unlike these earlier published accounts, it appears CMB-M0339 employs an NRPS-like aminoacylation enzyme with an adenylation domain tolerant of different amino acid substrates (i.e., Val, Leu, Ile, Pro, Ser, Thr, and homo-Ala).  A preliminary assessment of the noonindole biosynthetic gene cluster (BGC) suggests a biogenetic relationship linking 5-14 and inclusive of the minor co-metabolites i-viii starting with emindole SB (14) undergoing stereospecific epoxidation and ring closure to paspaline (11) followed by sequential oxidation to paspaline B (12) and the carboxylic acid 13, followed by decarboxylation and oxidation to paxilline (1) (Figure 9). Oxidation of 1 could then yield noonindole F (10) with further oxidation and/or amino acid acylation returning noonindoles A-E (5-9) and co-clustering minor analogues (iviii). Consistent with this hypothesis, close examination of the CMB-M0339 D400 extract GNPS and UPLC-DAD-MS data using single ion extraction (SIE) detected an ion with a molecular formula attributable to 1 ( Figure S71).   Table 3. Comparison of 5-9 and minor co-metabolites i-viii.
,c) Proposed hexacyclic scaffolds. Possible alternate isomers: A N-methyl-homoalanine, valine; B N-methyl-threonine; C pipecolic acid; D N,N-dimethyl-allo-isoleucine.   Our investigation into the marine-derived Aspergillus noonimiae CMB-M0339 led to the discovery of noonindoles A-F (5-10) and related minor analogues (i-viii) as new examples of a rare class of fungal indole diterpene amino acid conjugate. This discovery highlights the continued capacity of fungi to provide access to new chemical space and validates molecular networking (GNPS) as an effective platform to detect, dereplicate, and prioritize new over known chemistry, and cultivation profiling (MATRIX) as a means to optimise the production. Our discovery of the noonindoles suggests the CMB-M0339 features an NRPS-like aminoacyl modifying enzyme in the noonindole biosynthetic gene cluster (BGC) capable of accommodating and incorporating multiple lipophilic amino Our investigation into the marine-derived Aspergillus noonimiae CMB-M0339 led to the discovery of noonindoles A-F (5-10) and related minor analogues (i-viii) as new examples of a rare class of fungal indole diterpene amino acid conjugate. This discovery highlights the continued capacity of fungi to provide access to new chemical space and validates molecular networking (GNPS) as an effective platform to detect, dereplicate, and prioritize new over known chemistry, and cultivation profiling (MATRIX) as a means to optimise the production. Our discovery of the noonindoles suggests the CMB-M0339 features an NRPS-like aminoacyl modifying enzyme in the noonindole biosynthetic gene cluster (BGC) capable of accommodating and incorporating multiple lipophilic amino acids. Further studies into the structure, biosynthesis, and biology of these and other CMB-M0339 indole diterpenes are ongoing, and will be reported elsewhere.

General Experimental Procedures
Chemicals were purchased from Sigma-Aldrich or Merck unless otherwise specified. Solvent extractions were performed using analytical-grade solvents, while HPLC, UPLC, and HPLC-MS analyses employed HPLC-grade solvents supplied by Labscan or Sigma-Aldrich and filtered/degassed through 0.45 µm polytetrafluoroethylene (PTFE) membrane prior to use. Deuterated solvents were purchased from Cambridge Isotopes (Tewksbury, MA, USA). Microorganisms were manipulated under sterile conditions in a Laftech class II biological safety cabinet and incubated in either an MMM Friocell incubator (Lomb Scientific, NSW, Australia) or an Innova 42R incubator shaker (John Morris, NSW, Australia) at 26.5 • C. Semi-preparative and preparative HPLCs were performed using Agilent 1100 series HPLC instruments with corresponding detectors, fraction collectors, and software. Analytical UPLC chromatograms were obtained on an Agilent 1290 infinity UPLC instrument equipped with a diode array multiple wavelength detector (Zorbax C 8  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. Structural assignments were made with additional information from gCOSY, gHSQC, and gHMBC experiments.

Fungal Isolation and DNA Taxonomic Analysis
A marine sediment collected in 2008 from a location off Perth, Western Australia, was used to inoculate an M1 agar plate (inclusive of 3.3% artificial sea salt) which was incubated at 27 • C for 10-14 days, after which colony selection yielded an array of isolates including fungus CMB-M0339. Genomic DNA was extracted from the mycelia of CMB-M0339 using the DNeasy Plant Mini Kit (Qiagen) as per the manufacturers protocol, and the 18s rRNA genes were amplified by PCR using the universal primers ITS-1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS-4 (5 -TCCTCCGCTTATTGATATGC-3 ) purchased from Sigma-Aldrich. The PCR mixture (50 µL) containing 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 was amplified 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) and examined by agarose gel electrophoresis, with DNA sequencing performed by the Australian Genome Research Facility (AGRF) at The University of Queensland. A BLAST analysis (NCBI database) on the resulting CMB-M0339 ITS gene sequence ( Figures S1-S3, GenBank accession no. OP132523) revealed 92.5% identity with the fungal strain Aspergillus noonimiae.

Global Natural Product Social (GNPS) Molecular Networking
Aliquots (1 µL) of CMB-M0339 cultivation extract (100 µg/mL in MeOH) were analysed on an Agilent 6545 Q-TOF LC/MS equipped with an Agilent 1290 Infinity II UPLC system (Zorbax C 8 , 0.21 µm, 1.8 × 50 mm column, gradient elution at 0.417 mL/min over 2.5 min from 90% H 2 O/MeCN to MeCN with an isocratic 0.1% formic acid/MeCN modifier). UPLC-QTOF-(+) MS/MS data acquired for all samples at a collision energy of 35 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 [45] employing the spectral clustering algorithm with a cosine score of 0.5 and a minimum of 6 matched peaks. The resulting spectral network was imported into Cytoscape version 3.7.1 [47] and visualized using a ball-and-stick layout where nodes represent parent mass and cosine score was reflected by edge thickness. Moreover, group abundances were set as pie charts, which reflected the intensity of MS signals. MS/MS fragmentation analysis was performed on the same machine for ion detected in the full scan range at an intensity above 1000 counts at ten scans/s, with an isolation width of 4~m/z using fixed collision energy and a maximum of 3 selected precursors per cycle. General instrument parameters including gas temperature at 325 • C, drying gas 10 L/min, nebulizer 20 psig, sheath gas temperature 400 • C, fragmentation Volta 180eV, and skimmer 45 eV.

Scale Up Cultivation and Fractionation
The fungus CMB-M0339 was cultivated on D400 agar (×300 plates) at 27 • C for 10-14 days after which the agar and fungal mycelia were harvested and extracted with EtOAc (2 × 5 L), and the combined organic phase was filtered and concentrated in vacuo at 40 • C to yield an extract (2.9 g). This extract was sequentially triturated with n-hexane (20 mL), CH 2 Cl 2 (20 mL), MeOH (20 mL), and concentrated in vacuo to afford n-hexane   Phylogenetic tree obtained by PhyML Maximum Likelihood analysis was constructed using the top similar 18S rRNA sequences displayed after BLAST on Refseq RNA NCBI database using CMB-M0339 18S rRNA as queries ( Figure S4). The JC69 model was used to infer phylogeny sequences [48]. Sequence alignments were produced with the MUSCLE program [49]. Phylogenetic tree was constructed using the UGENE program using the aforementioned models and visualized using Ugene's tree view [50].

X-ray Crystallography
Crystals of 5 were obtained by slow evaporation from 50% DCM/Hexane in the cold room (−4 • C). Crystallographic data (Cu Kα, 2θ max = 125 • ) for 5 were collected on an Oxford Diffraction Gemini S Ultra CCD diffractometer with the crystal cooled to 190 K with an Oxford Cryosystems Desktop Cooler. Data reduction and empirical absorption corrections were carried out with the CrysAlisPro program. The structure was solved with SHELXT and refined with SHELXL [51]. The thermal ellipsoid diagrams were generated with Mercury [52]. All calculations were carried out within the WinGX graphical user interface [53]. The disordered water molecules in the structure were modelled with SQUEEZE implemented in PLATON [54]. The crystal data for 5 in CIF format were deposited in the CCDC database (2206901) (Table S13).

Antifungal Assay
The fungus Candida albicans ATCC 10231 was streaked onto a LB (Luria-Bertani) agar plate and was incubated at 37 • C for 48 h, after which a colony was transferred to fresh LB broth (15 mL) and the cell density was adjusted to 10 4 -10 5 CFU/mL. Test compounds were dissolved in DMSO and diluted with H 2 O to prepare 600 µM stock solutions (20% DMSO), which were serially diluted with 20% DMSO to provide concentrations from 600 µM to 0.2 µM in 20% DMSO. An aliquot (10 µL) of each dilution was transferred to a 96-well microtiter plate and freshly prepared fungal broth (190 µL) was added to prepare final concentrations of 30-0.01 µM in 1% DMSO. The plates were incubated at 27 • C for 48 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate (BMG LABTECH, Offenburg, Germany). Amphotericin B was used as the positive control (40 µg/mL in 10% DMSO). The IC 50 value was calculated as the concentration of the compound or antibiotic required for 50% inhibition of the bacterial cells using Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA). See Figure S80.

Antibacterial Assay
The bacterium to be tested was streaked onto an LB agar plate and was incubated at 37 • C for 24 h, after which a colony was transferred to fresh LB broth (15 mL) and the cell density was adjusted to 10 4-10 5 CFU/mL. Test compounds were dissolved in DMSO and diluted with H 2 O to give 600 µM stock solutions (20% DMSO), which were serially diluted with 20% DMSO to prepare concentrations from 600 µM to 0.2 µM in 20% DMSO. An aliquot (10 µL) of each dilution was transferred to a 96-well microtiter plate and freshly prepared microbial broth (190 µL) was added to provide final concentrations of 30-0.01 µM in 1% DMSO. The plates were incubated at 37 • C for 24 h and the optical density of each well was measured spectrophotometrically at 600 nm using POLARstar Omega plate (BMG LABTECH, Offenburg, Germany). Each test compound was screened against the Gram-negative bacterium Escherichia coli ATCC 11775 and the Gram-positive bacteria Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633. Rifampicin was used as the positive control (40 µg/mL in 10% DMSO) for Gram-positive bacteria and a mixture of rifampicin and ampicillin was used as the positive control for Gram-negative bacteria. The IC 50 value was calculated as the concentration of the compound or antibiotic required for 50% inhibition of the bacterial cells using Prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA). See Figure S80.

Cytotoxicity Assays
Human colorectal (SW620) and lung carcinoma (NCI-H460) cells were seeded evenly in a 96-well micro-plate (2000 cells/well in 180 µL of RPMI 1640 medium (Roswell Park Memorial Institute medium) supplemented with 10% FBS (Fetal Bovine Serum)) and the plate was incubated for 18 h (37 • C; 5% CO 2 ) to allow cells to attach. Test compounds were dissolved in 5% DMSO (v/v) and dilutions were generated from 300 µM to 300 nM. Aliquots (20 µL) of each dilution (or 5% aqueous DMSO for negative control and 5% aqueous SDS for positive control) were added to the plate in duplicate. After 68 h of incubation (37 • C; 5% CO 2 ), a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, USA) in PBS (Phosphate Buffered Saline) was added to each well to a final concentration of 0.4 mg/mL and plates were incubated for a further 4 h (37 • C; 5% CO 2 ) after which the medium was carefully aspirated and precipitated formazan crystals were dissolved in DMSO (100 µL/well). The absorbance of each well at 580 nm was measured with a PowerWave XS Microplate Reader from Bio-Tek Instruments Inc. (Vinooski, VT) and IC 50 values were calculated as the concentration of the compound required for 50% inhibition of the cancer cells using Prism 5.0 from GraphPad Software Inc. (La Jolla, CA, USA). See Figure S81.
Author Contributions: R.J.C. conceptualized the research; S.K. carried out the isolation, spectroscopic characterization, crystallization, and antibacterial, antifungal and cytotoxicity assays; P.V.B. performed the X-ray analyses; S.K. and Z.G.K. performed the taxonomic identification of the fungal strain; assigned molecular structures, and constructed the supplementary material; R.J.C. reviewed all data and drafted the manuscript, with support from S.K. and Z.G.K. All authors have read and agreed to the published version of the manuscript.