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Article

Molecular Networking and Cultivation Profiling Reveals Diverse Natural Product Classes from an Australian Soil-Derived Fungus Aspergillus sp. CMB-MRF324

by
Taizong Wu
1,
Angela A. Salim
1,
Paul V. Bernhardt
2 and
Robert J. Capon
1,*
1
Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
2
School of Chemistry and Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia
*
Author to whom correspondence should be addressed.
Molecules 2022, 27(24), 9066; https://doi.org/10.3390/molecules27249066
Submission received: 9 November 2022 / Revised: 16 December 2022 / Accepted: 16 December 2022 / Published: 19 December 2022
(This article belongs to the Special Issue Microbial Natural Products 2022)
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Versions Notes

Abstract

:
This study showcases the application of an integrated workflow of molecular networking chemical profiling (GNPS), together with miniaturized microbioreactor cultivation profiling (MATRIX) to successfully detect, dereplicate, prioritize, optimize the production, isolate, characterize, and identify a diverse selection of new chemically labile natural products from the Queensland sheep pasture soil-derived fungus Aspergillus sp. CMB-MRF324. More specifically, we report the new tryptamine enamino tripeptide aspergillamides E–F (7–8), dihydroquinoline-2-one aflaquinolones H–I (11–12), and prenylated phenylbutyrolactone aspulvinone Y (14), along with an array of known co-metabolites, including asterriquinones SU5228 (9) and CT5 (10), terrecyclic acid A (13), and aspulvinones N-CR (15), B (16), D (17), and H (18). Structure elucidation was achieved by a combination of detailed spectroscopic and chemical analysis, biosynthetic considerations, and in the case of 11, an X-ray crystallographic analysis.

1. Introduction

During our ongoing investigations into the chemistry of Australian microbes, we have reported numerous new structurally diverse natural products from many genera of fungi obtained from geographically dispersed and varying substrates. For example, our discoveries from the genus Aspergillus include a 2003 account of the depsi-cyclohexapeptide aspergillicins A–E from a Tasmanian estuarine sediment-derived Aspergillus carneus MST-MF156 [1]; a 2005 account of the heterocyclic dipeptide aspergillazines A–E from a remote outback Queensland soil-derived A. unilateralis MST-F8675 [2]; a 2009 account of the nor-methyl fumiquinazoline cottoquinazoline A and cyclopentapeptide cotteslosins A–B from a Western Australian beach sand-derived A. versicolor MST-MF495 [3]; a 2014 account of the P-glycoprotein inhibitory diketomorpholine shornephine A from a Queensland intertidal marine sediment-derived Aspergillus sp. CMB-M081F [4]; and a 2017 account of the nitro depsi-tetrapeptide diketopiperazine waspergillamide A from a Queensland mud dauber wasp-derived Aspergillus sp. CMB-W031 [5].
In recent years, these microbial biodiscovery efforts have been enhanced by the use of an integrated workflow consisting of global natural products social (GNPS) molecular networking [6] to detect, dereplicate, and prioritize, and a miniaturized microbioreactor methodology (MATRIX) [7] employing multiple different media under solid agar as well as static and shaken broth conditions to optimize the production of novel natural products. For example, in 2021 we reported a brown rice cultivation of a Queensland sheep pasture soil-derived Aspergillus sp. CMB-MRF324 yielding the acetylcholinesterase inhibitory meroterpene millmerranones A–F (1–6) (Figure 1), with millmerranone A being noteworthy as a rare example of a natural cyclic carbonate [8]. At that time, the GNPS analysis of a Sabouraud dextrose agar (SDA) cultivation of CMB-MRF324 revealed additional chemical diversity that was prioritized for future investigation.
This current report describes just such an investigation, where fractionation of the CMB-MRF324 SDA extract yielded new linear tripeptide aspergillamides E–F (7–8) as equilibrating E/Z amide rotamers incorporating a rare tryptamine enamine moiety, along with the known asterriquinones SU5228 (9) and CT5 (10) (Figure 1). In addition, we provide an account of a GNPS–MATRIX analysis of CMB-MRF324 that prompted discovery of the new dihydroquinoline-2-one aflaquinolones H (11) and I (12), the known terrecyclic acid A (13), and the new prenylated phenylbutyrolactone aspulvinone Y (14), and known aspulvinones N-CR (15), B (16), D (17) and H (18) (Figure 1). The detection, optimized production, isolation, and structure elucidation of 7–18 are outlined below.

2. Results and Discussion

A GNPS molecular network analysis of a selection of EtOAc extracts prepared from ×27 solid phase plate cultivations of pasture-derived fungi (SDA) and bacteria (ISP-2 agar) revealed two molecular families (clusters) unique to the fungus CMB-MRF324, both correlating with prominent peaks in the extract UPLC-DAD chromatogram (Figure 2A–C). Subsequent scaled-up SDA cultivation of CMB-MRF324 followed by extraction with EtOAc, solvent trituration and reversed phase chromatography (Figure S1) yielded the new tryptamine enamino tripeptide aspergillamides E–F (7–8) along with the known asterriquinones SU5228 (9) and CT5 (10) (Figure 1). Similarly, a GNPS based re-analysis of a brown rice cultivation of this same CMB-MRF324 fungus revealed a molecular family featuring potentially new chemistry, also evident in the UPLC-DAD chromatogram along with two other classes of metabolite (Figure 3A,B). Subsequent reversed phase and gel (LH-20) chromatography fractionation of a scaled up brown rice cultivation (Figure S2) yielded the new dihydroquinoline-2-one aflaquinolones H (11) and I (12) and prenylated phenylbutyrolactone aspulvinone Y (14), along with the known terrecyclic acid A (13) and aspulvinones N-CR (15), B (16), D (17), and H (18) (Figure 1). Structures for the known natural products 9 (Table S5 and Figures S19–S20) [9], 10 (Table S6 and Figures S21–S25) [10,11], 13 (Table S9 and Figures S40–S42) [12,13,14,15], 15 (Figures S48–S51) [16], 16 (Figures S52–S53) [17,18], 17 (Figures S54–S55) [17,18], and 18 (Figures S56–S57) [19] were established by spectroscopic analysis and comparison to data in the literature.
Of interest, although 10 possesses a plane-of-symmetry, this was not manifested in the 1D NMR data acquired in DMSO-d6 (Figure S21) or methanol-d4 (Figure S22), but was evident in the data acquired in CDCl3 (Figures S23 and S24). A possible explanation for this solvent effect (which has not been previously described) is that H-bonding in DMSO-d6 and methanol-d4 restricts/slows intramolecular rotations sufficiently to generate distinct and NMR detectable populations of atropisomeric stereoisomers, differentiating between a racemic mixture of P,P and M,M enantiomers on the one hand, and the meso P,M diastereomer on the other. As such, the apparent loss of symmetry in the NMR data is misleading and is perhaps better characterized as the detection of two separate isomer populations, each retaining a plane-of-symmetry. These phenomena suggest a need for caution when assessing the biological activity (and attempting any in silico modelling) of otherwise pure achiral (symmetric or asymmetric) compounds where H-bond mediated solvent effects (i.e., induced by aqueous assay media) can present as mixed populations of atropisomeric stereoisomers. An account of the structure elucidation of the new natural products 7–8, 11–12, and 14 is outlined below.
HRESI(+)MS measurement on 7 returned a molecular formula (C27H32N4O3, Δmmu −1.9) requiring 14 double bond equivalents (DBE). The 1D NMR (DMSO-d6) data for 7 (Table 1 and Table 2, Tables S1 and S2 and Figure 4 and Figures S5–S10) revealed an isomeric mixture (ratio 1:0.6). Analysis of NMR resonances attributed to the major isomer 7a revealed a mono substituted benzene, a C-2 mono substituted indole, an E 1,2-disubstituted double bond (J8,9 15 Hz), and three carbonyl carbons accounting for all DBE. Diagnostic 2D NMR correlations allowed assembly of the tripeptide scaffold for 7a, with ROESY correlations between the terminal N-acetyl-valine and associated N-methyl amide moiety establishing 7a as the E rotamer about the valine amide bond. Comparable analysis of the minor isomer 7b confirmed the same planar structure with ROESY correlations between the phenylalanine and valine, a methines consistent with the Z rotamer about the valine amide bond. A Marfey’s analysis performed on 7 confirmed incorporation of l-valine and N-methyl-l-phenylalanine residues (Figure S4), permitting assignment of the structure for aspergillamide E (7) as shown.
HRESI(+)MS measurement on 8 returned a molecular formula (C27H32N4O3, Δmmu +2.5) isomeric with 7. Comparison of the 1D and 2D NMR (DMSO-d6) data for 8 (Table 1 and Table 2, Tables S3 and S4 and Figure 4 and Figures S12–S17) with 7 revealed the same equilibrating major E (8a) and minor Z (8b) amide rotamers with key differences between 8 and 7 attributed to a Z enamino configuration (J8,9 9.5 Hz). A Marfey’s analysis performed on 8 confirmed the incorporation of l-valine and N-methyl-l-phenylalanine residues (Figure S4), permitting assignment of the structure for aspergillamide F (8) as shown. Alert to the potential for chemically reactive natural products to form handling artifacts [20], we determined that the Z geometric isomer 8 isomerizes on standing, under either acidic or neutral conditions, to the E isomer 7 and that this process is accelerated on heating. Nevertheless, chemical analysis of a fresh extract detected both 7 and 8, confirming their status as natural products.
Tryptamine enamino tripeptides have appeared in the natural products literature on several occasions over the last 25 years, starting in 1997 with terpeptin (19) from a soil-derived Aspergillus terreus 95F-1 collected near Naha City, Okinawa Prefecture, Japan [21]; and progressing through 1998 with aspergillamides A–B (20–21) from a saline lake sediment-derived Aspergillus sp. CNC-120 collected in the Bahamas [22]; 2002 with miyakamides A1, A2, B1, and B2 (22–25) from a fallen leaf-derived Aspergillus flavus Link var. columnaris FKI-0739 collected off Miyakojima Island, Japan [23]; 2008 with terpeptins A–B (26–27) from the mangrove plant (Acanthus ilicifolius) epiphyte Aspergillus sp. W–6 collected in Dongzhai Gang, China [24]; 2010 with JBIR-81 (28) and JBIR-82 (29) from the seaweed (Sargassum sp.) derived Aspergillus sp. SpD081030G1f1 collected off Ishigaki Island, Okinawa Prefecture, Japan [25]; and 2019 with aspergillamides C–D (30–31) from a marine sponge-derived Aspergillus terreus SCSIO 41008 collected off Guangdong Province, China [26] (Figure 5). It is noteworthy that all these reports were from fungi of the genus Aspergillus, albeit isolated from geographically diverse terrestrial and marine substrates. Encouraged by the relatively small number of reported members of this class of natural product, we returned our attention to the GNPS molecular network analysis and the MS/MS fragmentations that defined the aspergillamide molecular family. With diagnostic losses of 158 and 301 amu revealing the sodiated ion for the N-terminal amino acid, it was possible to detect nodes consistent with the known aspergillamides A–D (20–21, 30–31), and for putative new very minor homologues, homo-Ala (i) and Ala (ii) (Figure 6 and Figures S58–S61).
HRESI(+)MS measurement on 11 returned a molecular formula (C27H31NO6, Δmmu −2.0) requiring 13 DBE. The 1D NMR (CDCl3) data for 11 (Table 3 and Table S7, Figures S26–S31) revealed resonances for twelve sp2 carbons, of which eight could be attributed to two aromatic rings and four to an E 1,2-disubstituted double bond, an ester/amide, and a ketone carbonyl, accounting eleven DBE and requiring that 11 incorporate two additional rings. Diagnostic 2D NMR correlations permitted assembly of a planar structure (Figure 7) with a single crystal X-ray crystallographic analysis (Figure 8) unambiguously establishing the structure and absolute configuration of aflaquinolone H (11).
Remarkably, the crystal structure of 11 comprises six independent molecules in the asymmetric unit; molecule A is shown in Figure 8. All molecules adopt similar conformations with the only significant difference being the orientation of the 4-methoxy group on the phenyl ring which is found in one of two conformers related by a 180° rotation. An overlay of the six molecules is shown in the Supplementary Materials. A strong intramolecular H-bond (O5A-H…O4A 1.89 Å, 144.9°) corresponding to hydroxyl groups in the 4- and 6-positions of the 3, 4-dihydroquinolinone ring system is apparent in Figure 8, and this is present in all six molecules comprising the structure. This H-bond restrains the 4-hydroxy group (O4A) in an equatorial position, and this locks the confirmation of the bicyclic ring system leaving the p-methoxyphenyl substituent in an axial position. The absolute configuration of 11 was established by the Bijvoet analysis of Hooft and co-workers; P2 = 1.000, Hooft parameter y = −0.06(9) [27].
HRESI(+)MS measurement on 12 revealed a molecular formula (C27H33NO6, Δmmu −1.0), consistent with a hydrogenated analogue of 11. Comparison of the NMR (CDCl3) data for 12 (Table 3 and Table S8, Figures S33–S38) with 11 showed a high level of similarity, with differences attributed to replacement of the C-22 ketone in 11 (δH 214.0) with a C-22/H-22 2°-OH in 12 (δH 3.12, ddd, J = 10.5, 10.5, 4.5 Hz, H-22; δC 77.3, C-22), further confirmed by diagnostic 2D NMR correlations (Figure 7). The β-facial axial orientation of H-22 was evident from large values for J21,22 and J22,23a (10.5 and 10.5 Hz) which together with biogenetic considerations permitted assignment of the full structure for aflaquinolone I (12).
Aflaquinolones H–I (11–12) from Aspergillus sp. CMB-MRF324 bear a carbo/heterocyclic scaffold in common with a small number of known fungal metabolites: aflaquinolones A–D (32–35) reported in 2012 from the Hawaiian Aspergillus sp. NRRL 58570 and Korean Aspergillus sp. SF-5044 [28]; scopuquinolone B (36) reported in 2018 from a coral-derived Scopulariopsis sp. [29]; and 22-O-(N-methyl-L-valyl)aflaquinolone B (37) and 22-O-(N-methyl-L-valyl)-21-epi-aflaquinolone B (37) reported in 2014 from the South China Sea gorgonian-derived Aspergillus sp. XS-20090B15, as nM potent inhibitors of the respiratory syncytial virus (RSV) [30] (Figure 9).
HRESI(+)MS measurement on 14 returned a molecular formula (C27H28O6, Δmmu −2.4) suggestive of an oxidized (+O) analogue of the co-metabolite aspulvinone B (16). Comparison of the 1D NMR (methanol-d4) data for 14 (Table 4 and Table S10, Figures S43–S46) with the 1D NMR (acetone-d6) data for 16 (Figure S52, Note—16 proved insoluble in methanol) revealed a high level of similarity, with key differences attributed to replacement of the isoprene D8″,9″ moiety in 16 (dH 5.36, m, H-8″; δC 123.3, C-8″; δC 133.1, C-9″) with a dioxygenated moiety in 14 (δH 3.82, dd, H-8″; δC 69.4, C-8″; δC 78.3, C-9″). The latter resonances could in principle be attributed to three possible substructures (Figure 10) consisting of a dihydrobenzofuran, a dihydrobenzopyran, or an epoxide. The dihydrobenzofuran moiety (Figure 10a) is prominent among known aspulvinones (Figure S62) and features a consistent range of 13C NMR (methanol-d4) chemical shifts for C-8″ (δC 89.9–91.4) and C-9″ (δC 71.1–73.1) [16,31]. While the dihydrobenzopyran moiety (Figure 10b) is less common among known aspulvinones (Figure S62), there are nevertheless reports of the 13C NMR (methanol-d4) chemical shifts for C-8″ (δC 68.7–71.0) and C-9″ (δC 77.2–77.5) [16]. Finally, although there are no known accounts of aspulvinones featuring an epoxy moiety (Figure 10c), there are reports of 13C NMR chemical shifts that are relevant to both C-8″ (δC 60–65) and C-9″ (δC 57–59) [32]. These spectroscopic comparisons, together with diagnostic 2D NMR correlations (Figure 11), and biogenetic considerations, permitted assignment of the structure for aspulvinone Y (14) as shown. The sample of 14 proved unstable and decomposed during NMR data acquisition, precluding measurement of an optical rotation and consideration of absolute configuration.
A review of the scientific literature revealed a rich history of fungal natural products belonging to the aspulvinone structure class. While these originated with 1973 [17] and 1975 [18] accounts of the pulvinones from Aspergillus terreus IAM 2054, the trivial nomenclature and structures were subsequently revised in 1976 [19] and 1979 [33] as aspulvinones A–I. These early discoveries were followed by reports of new members of this structure class, including a 2011 account of aspulvinones I-CR to N-CR from a Costa Rican marine sediment-derived Aspergillus sp. 05545 [16]; a 2013 account of isoaspulvinone E from a Fijian marine mangrove rhizosphere soil-derived Aspergillus terreus Gwq-48 [34]; a 2013 account of aspulvinone B1 from Aspergillus terreus NIH 2624 [35]; a 2015 account of aspulvinone O from a Chinese marine red alga ephiphyte Paecilomyces variotii EN-291 [36] (curiously the trivial nomenclature “aspulvinones J–N” do not appear to have been utilised); a 2016 account of aspulvinone P–Q from a Chinese wetland mud-derived Aspergillus flavipes PJ03-11 [37]; a 2017 account of aspulvinone R from a Chinese medicinal plant-derived Aspergillus sp. CPCC 400735 [38]; a 2021 account of aspulvinones R–U from a Thai marine sponge-derived Aspergillus flavipes KUFA1152 [39] (note—the trivial nomenclature “aspulvinone R” had been prior assigned to a different fungal metabolite in 2017 [38]); a 2021 account of aspulvinones V-X from a marine-derived Aspergillus terreus ASM-1 [31]; a 2022 account of aspulvins A–H from the Chinese medicinal plant-derived Cladosporium sp. 7951 [40]; and a 2022 account of aspulvinones S–V from the Taiwanese marine green alga-derived Aspergillus terreus NTU243 [41] (note—the trivial nomenclature “aspulvinones S-V” had been prior assigned to different fungal metabolites in 2021 [31,39]). Given this history, we attributed the trivial nomenclature aspulvinone Y to 14.
Aspulvinone D (17) may be considered as the biosynthetic precursor of other aspulvinone analogs via a putative epoxide intermediate, which can be further modified (either enzymatically or non-enzymatically) [20] by one of three pathways: (i) intramolecular SN2 addition of the C-4″ phenol moiety to C-8″ with concomitant opening of the epoxide to yield aspulvinone F, (ii) intramolecular SN2 addition of the C-4″ phenol moiety to C-9″ with concomitant opening of the epoxide to yield aspulvinone N–CR (15), or (iii) SN2 addition of H2O to C-9″ (or C-8″) with concomitant opening of the epoxide to yield aspulvinone L–CR (Figure 12). This same set of transformations could in principle be applied to other (as yet undetected) aspulvinone/aspulvin epoxides as a pathway to all known heterocyclic members of this structure class.
In summary, an integrated workflow of chemical (GNPS) and cultivation (MATRIX) profiling has proved effective at exploring the chemical diversity encoded within the genome of the Queensland sheep pasture soil-derived fungus Aspergillus sp. CMB-MRF324. In addition to meroterpene millmeranones A–F (1–6), this latest study has demonstrated that CMB-MRF324 also produces new tryptamine enamino tripeptides, aspergillamide E–F (7–8), new dihydroquinoline-2-one aflaquinolones H–I (11–12), and a new prenylated phenylbutyrolactone aspulvinone Y (14), along with an array of known metabolites. The majority of the new natural products documented in this study proved chemically unstable once purified, further demonstrating the importance of employing effective and sensitive analytical methods to monitor and ensure chemical integrity during purification, characterisation, handling, and storage.

3. Materials and Methods

3.1. General Experimental Procedure

Chiroptical measurements ([α]D) were obtained on a JASCO P-1010 polarimeter (JASCO International Co. Ltd., Tokyo, Japan) in a 100 × 2 mm cell at specified temperatures. Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker Avance 600 MHz spectrometer (Bruker Pty. Ltd., Preston, VIC, Australia) with either a 5 mm PASEL 1H/D-13C Z-Gradient probe or 5 mm CPTCI 1H/19F-13C/15N/DZ-Gradient cryoprobe. In all cases, spectra were acquired at 25 °C in DMSO-d6 or CDCl3 or methanol-d4 or acetone-d6 with referencing to residual 1H or 13C signals (DMSO-d6, δH 2.50 and δC 39.5; CDCl3 δH 7.26 and δC 77.1; methanol-d4, δH 3.31 and δC 49.0; acetone-d6, δH 2.05). High-resolution ESIMS spectra were obtained on a Bruker micrOTOF mass spectrometer (Bruker Pty. Ltd., Preston, VIC, Australia) by direct injection in MeOH at 3 μL/min using sodium formate clusters as an internal calibrant. Liquid chromatography diode array mass spectrometry (HPLC-DAD-MS) data were acquired either on an Agilent 1260 infinity II separation module equipped with an Agilent G6125B LC/MSD mass detector (Agilent Technologies Pty. Ltd, Mulgrave, VIC, Australia) and diode array detector or on Shimadzu LCMS-2020 LCMS (Shimadzu Pty. Ltd., Rydalmere, NSW, Australia). Semi-preparative HPLC were performed using Agilent 1100 series HPLC instruments with corresponding detectors, fraction collectors and software. UPLC chromatograms were obtained on Agilent 1290 infinity UPLC system equipped with diode array multiple wavelength detector (Agilent Zorbax C8 RRHD 1.8 μm, 50 × 2.1 mm column (Agilent Technologies Pty. Ltd., Mulgrave, VIC, Australia), gradient elution over 2.50 min at 0.417 mL/min from 90% H2O/MeCN to 100% MeCN with an isocratic 0.01% TFA modifier). UPLC-QTOF analysis was performed on an Agilent 1290 Infinity II UPLC (Agilent Zorbax C8 RRHD 1.8 μm, 50 × 2.1 mm column, gradient elution over 2.50 min at 0.417 mL/min from 90% H2O/MeCN to 100% MeCN with an isocratic 0.1% formic acid/MeCN modifier) coupled to an Agilent 6545 Q-TOF (Agilent Technologies Pty. Ltd, Mulgrave, VIC, Australia). 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. Chemicals were purchased from Sigma-Aldrich or Merck unless otherwise specified. Analytical grade solvents were used for solvent extractions. Chromatography solvents were of HPLC grade 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. Microorganisms were manipulated under sterile conditions using a Laftech class II biological safety cabinet and incubated in either MMM Friocell incubators (Lomb Scientific Pty. Ltd., Taren Point, NSW, Australia) or an Innova 42R incubator shaker (John Morris Scientific Pty. Ltd., Chatswood, NSW, Australia).

3.2. Collection and Taxonomy of CMB-MRF324

The fungus CMB-MRF324 was isolated from the soil sample collected from a sheep pasture near Millmerran, Queensland, Australia. Following 7 days cultivation on an SD agar plate at 30 °C., Genomic DNA was extracted from the mycelia using the DNeasy Plant Mini Kit (Qiagen, Clayton, VIC, Australia), the 18S rRNA genes were amplified, and BLAST analysis (NCBI database) confirmed the closest homology with Aspergillus terreus (Genbank accession number: MZ823609) as previously reported [8].

3.3. Scale-Up SDA Cultivation and Fractionation of CMB-MRF324

CMB-MRF324 was incubated at 30 °C for 14 days on ×20 SDA plates after which the combined agar and mycelia was harvested and extracted with EtOAc (2 × 300 mL) and the organic phase concentrated in vacuo to yield an extract (732 mg). The extract was then subjected to sequential trituration to afford (after drying under nitrogen at 40 °C) n-hexane (380 mg) and MeOH (324 mg) solubles, with the latter subjected to preparative reverse-phase HPLC (Phenomenex Luna-C8 10 μm, 21.2 × 250 mm column (Phenomenex Inc., Lane Cove, NSW, Australia), 20 mL/min gradient elution over 25 min from 60% H2O/MeCN to 15% H2O/MeCN with an isocratic 0.01% TFA/MeCN modifier) to yield fractions F1–F2. Fraction F1 was then subjected to semi-preparative HPLC (Agilent Zorbax C8 5 μm, 250 × 9.4 mm column (Agilent Technologies Pty. Ltd., Mulgrave, VIC, Australia), 3 mL/min isocratic elution over 20 min at 55% H2O/MeCN to yield 7 (7.0 mg, 0.9%) and 8 (4.0 mg, 0.5%), while Fraction F2 was subjected to semi-preparative HPLC (Agilent Zorbax Eclipse XDB C8 5 μm, 250 × 9.4 mm column (Agilent Technologies Pty. Ltd., Mulgrave, VIC, Australia), 3 mL/min gradient elution over 15 min from 40% H2O/MeCN to 25% H2O/MeCN, with an isocratic 0.01% TFA/MeCN modifier) to yield 9 (5.0 mg, 0.7%) and 10 (5.0 mg, 0.7%). (Note: All % yields are weight to weight estimates based on unfractionated EtOAc extract).

3.4. Scale-Up Brown Rice Cultivation and Fractionation of CMB-MRF324

CMB-MRF324 was incubated at room temperature for 24 days in rice-based medium (70 g brown rice, 0.3 g peptone, 0.3 g yeast extract, 0.1 g monosodium glutamate, in 100 mL distilled H2O) after which the medium and mycelia were extracted with EtOAc (3 × 500 mL) and the organic phase concentrated in vacuo to yield an extract (3.8 g). The extract was then subjected to sequential trituration to afford (after drying in vacuo) n-hexane, CH2Cl2 (3.05 g), and MeOH solubles. A portion of the CH2Cl2 solubles (2.4 g) was subjected to preparative HPLC (Phenomenex Luna-C8 10 μm, 21.2 × 250 mm column, 20 mL/min gradient elution over 30 min from 90% H2O/MeCN to 100% MeCN with an isocratic 0.01% trifluoroacetic acid/MeCN modifier) to yield 40 fractions (F1–F40). F18 (50 mg) was subjected to semi-preparative HPLC (Agilent Zorbax SB C3,5 μm, 9.4 × 250 mm column, 3 mL/min gradient elution over 20 min from 60% MeCN/H2O to 55% MeCN/H2O with an isocratic 0.01% TFA/MeCN modifier) to yield 13 (4.0 mg, 0.1%). Combined F21–F24 (230 mg) were subjected to gel chromatography (Sephadex LH-20 (Sigma-Aldrich Pty. Ltd, Macquarie Park, NSW, Australia) gravity elution with 50% MeOH/CH2Cl2) followed by semi-preparative HPLC (Agilent Zorbax SB C3 5 μm, 9.4 × 250 mm column, 3 mL/min isocratic elution over 25 min with 55% H2O/MeCN with an isocratic 0.01% TFA/MeCN modifier) to yield 11 (5.0 mg, 0.2%) and 12 (1.6 mg, 0.06%). F25 was subjected to semi-preparative HPLC (Agilent Zorbax Eclipse XDB C8 5 μm, 9.4 × 250 mm column, 3 mL/min isocratic elution over 20 min with 45% H2O/MeCN with an isocratic 0.01% TFA/MeCN modifier) to yield 14 (1.0 mg, 0.03%), 15 (1.0 mg, 0.03%), and 16 (3.0 mg, 0.09%). F31 subjected to semi-preparative HPLC (Agilent Zorbax Eclipse XDB C8 5 μm, 9.4 × 250 mm column, 3 mL/min gradient elution over 20 min from 40% H2O/MeCN to 35% H2O/MeCN with an isocratic 0.01% TFA/MeCN modifier) to yield 17 (2.5 mg, 0.07%) and 18 (2.5 mg, 0.07%). (Note: All % yields are weight to weight estimates based on unfractionated EtOAc extract).

3.5. Metabolite Characterization

Aspergillamide E (7). White powder; NMR (600 MHz, DMSO-d6), Table 1, Table 2, Tables S1 and S2, Figures S5–S10; HRESI(+)MS m/z 483.2348 [M + Na]+ (calcd for C27H32N4NaO3 483.2367).
Aspergillamide F (8). white powder; 1D and 2D NMR (DMSO-d6), Table 1, Table 2, Tables S3 and S4, Figures S12–S17; HRESI(+)MS m/z 483.2392 [M + Na]+ (calcd for C27H32N4NaO3 483.2367).
Asterriquinone SU5228 (9). 1D NMR (DMSO-d6), Table S5, Figure S19; HRESI(+)MS m/z 461.1475 [M + Na]+ (calcd for C27H22NaN2O4 461.1472).
Asterriquinone CT5 (10). 1D NMR (DMSO-d6), Table S6, Figures S21–S24; HRESI(+)MS m/z 507.2282 [M + H]+ (calcd for C32H31N2O4 507.2278).
Aflaquinolone H (11). colorless crystal; [α]D +55 (c 0.1, MeOH); 1D and 2D NMR (CDCl3), Table 3 and Table S7, Figures S26–S31; HRESI(+)MS m/z 488.2044 [M + H]+ (calcd for C27H31NO6 488.2064).
Aflaquinolone I (12). White powder; [α]D +16 (c 0.1, MeOH); 1D and 2D NMR (CDCl3), Table 3 and Table S8, Figures S33–S38; HRESI(+)MS m/z 490.2200 [M + H]+ (calcd for C27H33NO6 490.2210).
Terrecyclic acid A (13) [14]. [α]D -57 (c 0.1, CHCl3); 1D NMR (DMSO-d6), Table S9, Figures S40–S41; HRESI(+)MS m/z 249.1506 [M + H]+ (calcd for C15H21O3 249.1485).
Aspulvinone Y (14). Yellowish powder; 1D and 2D NMR (methanol-d4), Table 4 and Table S10, Figures S43–S46; HRESI(+)MS m/z 449.1959 [M + H]+ (calcd for C27H29O6 449.1983).
Aspulvinone N-CR (15). Yellowish powder; 1D and 2D NMR (acetone-d6), Table 4 and Table S11, Figures S48–S50; HRESI(+)MS m/z 465.1908 [M + H]+ (calcd for C27H29O7 465.1917).
Aspulvinone B (16) [42]. A 1D NMR (acetone-d6), Figure S52; HRESI(+)MS m/z 455.1828 [M + Na]+ (calcd for C27H28NaO5 455.1829).
Aspulvinone D (17) [42]. A 1D NMR (acetone-d6), Figure S54; HRESI(+)MS m/z 449.1969 [M + H]+ (calcd for C27H29O6 449.1959).
Aspulvinone H (18) [35,43]. A 1D NMR (methanol-d4), Figure S56; HRESI(+)MS m/z 433.2029 [M + H]+ (calcd for C27H29O5 433.2010).

3.6. Marfey’s Analysis of Aspergillamides E (7) and F (8)

A sample of analyte (i.e., 7 or 8) (100 μg) in 6M HCl (200 μL) was heated overnight at 110 °C after which the sample was concentrated to dryness under N2 and the residue treated with 1N NaHCO3 (20 μL), 1% (w/v) 1-fluoro-2,4-dinitro-5-l-alanine amide (l-FDAA) in acetone (50 μL) at 40 °C for 60 min, followed by the addition of 1N HCl (20 μL) and dilution with MeCN prior to HPLC-MS analysis. Samples of the authentic amino acid standards l-valine and N-methyl-l-phenylalanine were subjected to the same derivatisation process with both l-FDAA and d-FDAA. Aliquots (5 μL) of the FDAA derivatised analytes and standards were subjected to HPLC-DAD-MS (Agilent Poroshell EC-C8 2.7 μm, 3.0 × 150 mm, 0.8 mL/min isocratic elution over 12 min with 70% H2O/MeCN with an isocratic 0.05% formic acid modifier) to confirm that 7 and 8 incorporated l-valine and N-methyl-l-phenylalanine (Figure S4).

3.7. Isomerisation of Aspergillamide F (8) to E (7)

Three samples A-C of 8 (10 μg in 100 μL MeOH each) were prepared. Sample A was stirred at room temperature for 24 h. Sample B was treated with 0.1% TFA at room temperature for 24 h. Sample C was heated at 60 °C for 24 h. After 24 h aliquots (5 μL) of each were analysed by UPLC-DAD (Zorbax C8 RRHD 1.8 μm, 50 × 2.1 mm column, gradient elution over 2.50 min at 0.417 mL/min from 90% H2O/MeCN to 100% MeCN with an isocratic 0.01% TFA modifier) to reveal that 8 transformed to 7 slowly at room temperature even in the presence of acid, while heating at 60 °C accelerated this conversion (Figure S3).

3.8. X-ray Crystallography

Crystals of 11 were obtained by slow evaporation from 50% MeOH/CH2Cl2 at room temperature. Crystallographic data (CuKα radiation 1.54184 Å, 2θmax = 125°) 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 (Oxford Diffraction vers. 171.38.46). The structure was solved by direct methods with SHELXT and refined with SHELXL [44]. The thermal ellipsoid diagrams were generated with Mercury [45]. All crystallographic calculations were carried out within the WinGX graphical user interface [46]. The crystal structures data in CIF format have been deposited in the Cambridge Structural Database (CCDC 2217840) (see Figure 8 and Supplementary Materials).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27249066/s1, Figures S1–S62 and Tables S1–S11 NMR spectra, tabulated NMR data and X-ray crystallographic data.

Author Contributions

Conceptualization, R.J.C.; GNPS analyses, isolation and characterization of metabolites: T.W.; X-ray crystallography and data analysis: P.V.B.; Supplementary Materials document: T.W.; whole data revision and writing—original draft preparation: R.J.C. with support from T.W. and A.A.S.; writing—review and editing: all authors. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded in part by the Institute for Molecular Bioscience, The University of Queensland.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Raw NMR data have been deposited in NP-MRD (The Natural Products Magnetic Resonance Database).

Acknowledgments

We thank Kaumadi Samarasekera for isolation of Aspergillus sp. CMB-MRF324. T.W. acknowledges The University of Queensland for an International Postgraduate Scholarship.

Conflicts of Interest

The authors declare that they have no conflict of interest.

Sample Availability

Samples of selected compounds may be available from the authors on request, subject to an approved Materials Transfer Agreement.

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Molecules 27 09066 g001 550
Figure 1. Natural products from Aspergillus sp. CMB-MRF324.
Figure 1. Natural products from Aspergillus sp. CMB-MRF324.
Molecules 27 09066 g001
Molecules 27 09066 g002 550
Figure 2. (A) UPLC-DAD (210 nm) chromatogram of the CMB-MRF324 SDA extract with UV-vis profiles for two key structure classes, and GNPS molecular families (clusters) for (B) aspergillamides and (C) asterriquinones. Highlights; aspergillamides (yellow); asterriquinones (green).
Figure 2. (A) UPLC-DAD (210 nm) chromatogram of the CMB-MRF324 SDA extract with UV-vis profiles for two key structure classes, and GNPS molecular families (clusters) for (B) aspergillamides and (C) asterriquinones. Highlights; aspergillamides (yellow); asterriquinones (green).
Molecules 27 09066 g002
Molecules 27 09066 g003 550
Figure 3. (A) UPLC-DAD (210 nm) chromatogram of an extract of a CMB-MRF324 brown rice cultivation with UV-vis profiles for three key structure classes and GNPS molecular families (clusters) for (B) aspulvinones. Highlights: aflaquinolones (yellow); terrecyclic acid A (green); aspulvinones (blue). Note—aflaquinolones and terrecyclic acid A do not form obvious GNPS molecular families.
Figure 3. (A) UPLC-DAD (210 nm) chromatogram of an extract of a CMB-MRF324 brown rice cultivation with UV-vis profiles for three key structure classes and GNPS molecular families (clusters) for (B) aspulvinones. Highlights: aflaquinolones (yellow); terrecyclic acid A (green); aspulvinones (blue). Note—aflaquinolones and terrecyclic acid A do not form obvious GNPS molecular families.
Molecules 27 09066 g003
Molecules 27 09066 g004 550
Figure 4. Selected 2D NMR (DMSO-d6) correlations for equilibrating E (7a and 8a) and Z (7b and 8b) amide rotamers of aspergillamides E–F (7–8).
Figure 4. Selected 2D NMR (DMSO-d6) correlations for equilibrating E (7a and 8a) and Z (7b and 8b) amide rotamers of aspergillamides E–F (7–8).
Molecules 27 09066 g004
Molecules 27 09066 g005 550
Figure 5. Known tryptamine enamino tripeptide natural products 19–31. Highlighted: tryptamine enamino tripeptide scaffold (green).
Figure 5. Known tryptamine enamino tripeptide natural products 19–31. Highlighted: tryptamine enamino tripeptide scaffold (green).
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Molecules 27 09066 g006 550
Figure 6. MS/MS fragmentation for nodes in the aspergillamide GNPS molecular family, revealing major aspergillamides E–F (7–8), possible minor known co-metabolite aspergillamides A–D (20–21, 30–31) and putative new homologues, homo-Ala (i) and Ala (ii).
Figure 6. MS/MS fragmentation for nodes in the aspergillamide GNPS molecular family, revealing major aspergillamides E–F (7–8), possible minor known co-metabolite aspergillamides A–D (20–21, 30–31) and putative new homologues, homo-Ala (i) and Ala (ii).
Molecules 27 09066 g006
Molecules 27 09066 g007 550
Figure 7. Selected 2D NMR (CDCl3) correlations for 11 and 12.
Figure 7. Selected 2D NMR (CDCl3) correlations for 11 and 12.
Molecules 27 09066 g007
Molecules 27 09066 g008 550
Figure 8. ORTEP view of the crystal structure of 11 (30% probability ellipsoids).
Figure 8. ORTEP view of the crystal structure of 11 (30% probability ellipsoids).
Molecules 27 09066 g008
Molecules 27 09066 g009 550
Figure 9. Structures for known aflaquinolones and analogues 32–38. Highlighted: carbo/heterocyclic scaffold (green).
Figure 9. Structures for known aflaquinolones and analogues 32–38. Highlighted: carbo/heterocyclic scaffold (green).
Molecules 27 09066 g009
Molecules 27 09066 g010 550
Figure 10. Isomeric substructures featuring a substituted (a) dihydrobenzofuran, (b) dihydrobenzopyran, and (c) epoxide. Highlight (blue) indicated key oxygenated residues.
Figure 10. Isomeric substructures featuring a substituted (a) dihydrobenzofuran, (b) dihydrobenzopyran, and (c) epoxide. Highlight (blue) indicated key oxygenated residues.
Molecules 27 09066 g010
Molecules 27 09066 g011 550
Figure 11. Selected 2D NMR (methanol-d4) correlations for 14.
Figure 11. Selected 2D NMR (methanol-d4) correlations for 14.
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Molecules 27 09066 g012 550
Figure 12. A plausible biogenetic relationship linking prenylated to hydroxylated and heterocyclic aspulvinones (i–iii three different pathways to yield various aspulvinone analogs). Highlighted (bold, yellow, blue, red, green) indicate biogenetically related residues.
Figure 12. A plausible biogenetic relationship linking prenylated to hydroxylated and heterocyclic aspulvinones (i–iii three different pathways to yield various aspulvinone analogs). Highlighted (bold, yellow, blue, red, green) indicate biogenetically related residues.
Molecules 27 09066 g012
Table
Table 1. 1H NMR (DMSO-d6) Data for Aspergillamides E–F (7–8).
Table 1. 1H NMR (DMSO-d6) Data for Aspergillamides E–F (7–8).
Position7a δH, Mult (J in Hz)7b δH, Mult (J in Hz)8a δH, Mult (J in Hz)8b δH, Mult (J in Hz)
enamino-Trp
27.42, d (2.2)7.45, d (2.2)7.53, d (2.3)7.71, d (2.3)
47.61, d (7.7)7.62, d (7.7)7.57, d (7.9)7.57 B
57.07, dd (7.7, 7.7)7.11, m7.02, dd (7.9, 7.3)7.03 B
67.12, m7.07, m7.12, dd (7.9, 7.3)7.12 B
77.38, d (8.0)7.36, d (8.0)7.38, d (7.9)7.37, d (8.3)
86.42, d (15.0)6.52, d (15.0)5.95, d (9.5)5.99, d (9.5)
97.27, dd (15.0, 9.9)7.32, dd (15.0, 9.9)6.64, dd (10.2, 9.5)6.61, dd (9.8, 9.5)
1-NH11.1, d (2.2)11.1, d (2.2)11.29, br s11.30, br s
9-NH9.93, d (9.9)9.92, d (9.9)9.01, d (10.2)9.23, d (9.8)
N-Me-l-Phe
25.27, dd (9.6, 6.1)4.98, dd (9.6, 5.2)5.53, dd (9.9, 6.0)5.30, dd (8.8, 6.0)
3a. 3.23, dd (14.3, 6.1)a. 3.32 Aa. 3.23, dd (15.0, 6.0)a. 3.37 A
b. 2.97, dd (14.3, 9.6)b. 2.95, d (14.3, 6.1)b. 3.00, dd (15.0, 9.9)b. 2.96, dd (14.4, 8.8)
57.21, d (7.1)7.29, d (7.1)7.23, d (7.1)7.30, d (7.0)
67.24, dd (7.1, 7.1)7.30, dd (7.1, 7. 1)7.22, dd (7.1, 7.1)7.29, dd (7.0, 7.0)
77.18, t (7.1)7.22, t (7.1)7.17, t (7.1)7.22 B
N-Me3.08, s2.81, s3.03, s2.89, s
l-Val
24.44, dd (8.7, 8.7)4.29, dd (7.6, 7.6)4.46, dd (8.9, 8.9)4.26, dd (8.1, 8.1)
31.90, dqq (8.7, 6.9, 6.9)1.23, dqq (7.6, 6.9, 6.9)1.89, dqq (8.9, 6.7, 6.7)1.56, dqq (8.1, 6.8, 6.8)
4C0.81, d (6.9)0.63, d (6.9)0.72, d (6.7)0.48, d (6.8)
5C0.84, d (6.9)0.48, d (6.9)0.77, d (6.7)0.69, d (6.8)
NH7.97, d (8.7)8.26, d (7.6)7.9, d (8.9)8.24, d (8.1)
NHAc1.79, s1.91, s1.73, s1.66, s
A Resonance obscured by residual H2O and detected by HSQC; B E/Z amide rotamer resonances overlapping.
Table
Table 2. 13C NMR (DMSO-d6) Data for aspergillamides E–F (7–8).
Table 2. 13C NMR (DMSO-d6) Data for aspergillamides E–F (7–8).
Position7a δC, Type7b δC, Type8a δC, Type8b δC, Type
enamino-Trp
2123.5, CH123.6, CH123.6, CH124.4, CH
3111.5, C111.4, C109.5, C109.2, C
3a124.8, C124.9, C126.5, C126.6, C
4118.9, CH118.8, CH118.3, CH118.3, CH
5119.3, CH119.3, CH119.1, CH119.1, CH
6121.4, CH121.4, CH121.6, CH121.5, CH
7111.8, CH111.8, CH111.5, CH111.4, CH
7a136.8, C136.8, C135.6, C135.6, C
8106.8, CH107.0, CH103.3, CH104.2, CH
9119.5, CH119.5, CH118.1, CH117.9, CH
N-Me-l-Phe
1167.0, C166.1, C168.4, C167.6, C
257.7, CH61.3, CH56.8, CH60.6, CH
334.1, CH234.2, CH233.5, CH235.4, CH2
4137.6, C137.8, C137.4, C137.5, C
5128.8, CH129.3, CH128.8, CH129.3, CH
6128.1, CH128.4, CH128.1, CH128.3, CH
7126.3, CH126.5, CH126.3, CH126.5, CH
N-Me32.3, CH329.3, CH331.6, CH329.7, CH3
l-Val
1172.4, C171.5, C172.8, C171.8, C
254.0, CH53.6, CH53.6, CH54.0, CH
330.0, CH28.7, CH30.0, CH29.2, CH
4 A18.4, CH317.5, CH318.9, CH319.3, CH3
5 A18.9, CH319.3, CH318.2, CH318.0, CH3
NHCOCH3169.1, C170.6, C168.8, C170.2, C
NHCOCH322.2, CH322.2, CH322.1, CH321.7, CH3
A Assignments with same superscript are interchangeable.
Table
Table 3. 1D NMR (600 MHz, CDCl3) data for aflaquinolones H–I (11–12).
Table 3. 1D NMR (600 MHz, CDCl3) data for aflaquinolones H–I (11–12).
Position1112
δH, Mult (J in Hz)δC, TypeδH, Mult (J in Hz)δC, Type
2-165.8, C-165.4, C
33.70, br d (1.5)84.3, CH3.69, br d (1.4)84.4, CH
3-OMe3.61, s59.1, C3.60, s59.0, C
4-78.9, C-78.9, C
5-111.1, C-111.0, C
6-155.2, C-155.0, C
7-122.4, C-123.0, C
87.40, d (8.2)127.3, CH7.37, d (8.1)127.1, CH
96.36, d (8.2)107.0, CH6.32, d (8.1)106.8, CH
10-134.4, C-134.0, C
11-129.2, C-129.2, C
127.18, d (8.8)128.0, CH7.18, d (8.8)128.0, CH
136.82, d (8.8)114.5, CH6.83, d (8.8)114.5, CH
14-160.5, C-160.4, C
14-OMe3.76, s55.4, CH33.76, s55.5, CH3
156.82, d (8.8)114.5, CH6.83, d (8.8)114.5, CH
167.18, d (8.8)128.0, CH7.18, d (8.8)128.0, CH
176.79, d (16.6)122.7, CH6.61, d (16.8)121.8, CH
186.28, d (16.6)135.9, CH6.14, d (16.8)137.7, CH
19-37.6, C-37.2, C
20a. 2.11, m47.7, CH2a. 1.74, m46.1, CH2
b. 1.47, dd (13.4, 13.4) b. 1.07, m
212.54, m41.5, CH1.57 A36.3, CH
22-214.0, C3.12, ddd (10.5, 10.5, 4.5)77.3, CH
23a. 2.49, dd (14.2, 5.7)38.7, CH2a. 1.77, m32.1, CH2
b. 2.24, m b. 1.51 A
24a. 2.15, m38.5, CH2a. 1.82, m36.9, CH2
b. 1.73, td (13.4, 4.4) b. 1.37, td (13.7, 3.5)
251.10, s30.6, CH31.03, s31.3, CH3
260.99, d (6.5)14.6, CH30.98, d (6.4)18.8, CH3
1-NH7.75, br s-7.35, br s-
4-OH4.58, s-4.54, s-
6-OH9.14, s-9.08, s-
A Resonances obscured by solvent, observed by HSQC.
Table
Table 4. 1D NMR (methanol-d4) data for aspulvinone Y (14).
Table 4. 1D NMR (methanol-d4) data for aspulvinone Y (14).
Position14
δH, Mult (J in Hz)δC, Type
1-ND
2-101.6
3-164.2
4-142.3
56.38, s108.0
1′-122.6
2′7.64, d (1.5)129.7
3′-121.7
4′-154.3
5′6.73, d (8.3)117.6
6′7.62, dd (8.3, 1.5)127.7
7′2.83, t (6.8)23.1
8′1.84, t (6.8)33.5
9′-75.2
10′1.33, s26.8
11′1.33, s26.8
1″-126.3
2″7.57, s133.1
3″-121.4
4″-155.0
5″6.78, d (8.6)118.1
6″7.51, d (1.5)130.9
7″a. 3.07, dd (16.4, 5.0)31.8
b. 2.78, dd (16.4, 7.3)
8″3.79, dd (7.3, 5.0)69.9
9″-78.5
10″1.29, s21.1
11″1.35, s25.6
ND: Not detected due to low intensity.
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Wu, T.; Salim, A.A.; Bernhardt, P.V.; Capon, R.J. Molecular Networking and Cultivation Profiling Reveals Diverse Natural Product Classes from an Australian Soil-Derived Fungus Aspergillus sp. CMB-MRF324. Molecules 2022, 27, 9066. https://doi.org/10.3390/molecules27249066

AMA Style

Wu T, Salim AA, Bernhardt PV, Capon RJ. Molecular Networking and Cultivation Profiling Reveals Diverse Natural Product Classes from an Australian Soil-Derived Fungus Aspergillus sp. CMB-MRF324. Molecules. 2022; 27(24):9066. https://doi.org/10.3390/molecules27249066

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Wu, Taizong, Angela A. Salim, Paul V. Bernhardt, and Robert J. Capon. 2022. "Molecular Networking and Cultivation Profiling Reveals Diverse Natural Product Classes from an Australian Soil-Derived Fungus Aspergillus sp. CMB-MRF324" Molecules 27, no. 24: 9066. https://doi.org/10.3390/molecules27249066

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