Sulochrins and alkaloids from a fennel endophyte Aspergillus sp. FVL2

Abstract The fungal endophyte Aspergillus sp. strain FVL2, isolated from the traditional medicinal fennel plant, Foeniculum vulgare, was investigated for secondary metabolites. Fermentation on rice medium followed by chromatographic separation delivered three new natural products, 7-demethyl-neosulochrin (1), fumigaclavine I (3) and N-benzoyl-tryptophan (6) together with further 14 known metabolites, 1-O-methyl-sulochrin-4'-sulfate, questin, laccaic acid, isorhodoptilometrin, fumigaclavine A, fumigaclavine C, fumitremorgin C, fumigaquinazoline C, tryptoquivaline J, trypacidin, 3'-O-demethyl-sulochrin, 1-O-methyl-sulochrin, protocatechuic acid, and vermelone. The chemical structures of the new metabolites were determined by NMR spectroscopy and ESI HR mass spectrometry. For fumigaclavine I, we observed the partial deuterium transfer from the solvent to the enol form with a remarkable high stereo selectivity. The discovery of the new seco-anthraquinone 7-demethyl-neosulochrin (1) revealed a second type of ring cleavage by a questin oxygenase. The discovery of diverse secondary metabolites broadens the chemical space of Aspergillus. Graphical Abstract


Introduction
Aspergillus is a fungal genus with actually more than 400 species (Cole et al. 1977), some of them having a long history of use in the food fermentation industry, e.g., A. oryzae (Park et al. 2017). While some species are producers of important drugs, e.g., lovastatin from A. terreus (McKenney 1988), others are notorious human pathogens and produce harmful mycotoxins, e.g., the fumitremorgins from A. fumigatus (Latg e 1999) or the aflatoxins from A. flavus (Cole and Cox 1981). Like many other microorganisms, some A. spp. are endophytes, residing in the healthy tissue of different plants. Recent evidence showed that Aspergillus endophytes represent an enormous source of secondary metabolites, producing butenolides, alkaloids, terpenoids, cytochalasins, phenalenones, p-terphenyls, xanthones, sterols, diphenyl ethers, and anthraquinone derivatives (El-Hawary et al. 2020). Foeniculum vulgare (Fennel) is used as a vegetable and a traditional medicinal plant with antioxidative, anti-inflammatory, estrogenic, diuretic, gynecological, antithrombotic, and antihypertensive features (Rahimi et al. 2012). Despite the previous characterization of the bioactive compounds from this plant (Mhaidat et al. 2014;Kooti et al. 2015), chemical investigation of its fungal endophytes remained unexplored.

Structure elucidation
To characterize these metabolites, the fungus was cultured on 300 g solid rice medium. The ethyl acetate extract was subjected to silica gel column chromatography, followed by separation on Sephadex LH-20 and preparative HPLC, yielding seventeen compounds including three new metabolites 1 (2 mg), 3 (1 mg) and 6 (1 mg), which were studied by NMR spectroscopy (Tables S1-S3) and HR mass spectrometry.
Compound 1 was obtained as a colorless solid with moderate polarity, showing UV absorbance and a faint orange color reaction with anisaldehyde/sulfuric acid. HRMS indicated a molecular formula of C 16 H 14 O 7. The presence of signals for four aromatic meta-coupled methine protons in the 1 H NMR spectrum indicated the presence of two aromatic systems (rings A, B, Figure 1). Moreover, signals for one ether-/ester-linked methyl (d 3.32) and an aromatic ring-bound methyl (d 2.32) were also observed. The 13 C NMR spectrum revealed 10 fully substituted sp 2 carbon atoms including one carbonyl (d 201.7), one acid or ester carbonyl (d 171.5), and four phenolic carbon atoms between d 150 and 170 ppm. According to the COSY/HSQC experiments ( Figure S2, Table S1), the aromatic ring attached methyl group (d 2.32, d 21.3) is flanked by the meta-coupled protons at d 7.28 and 6.78, while the other meta-coupled protons (d 5.90, 5.78) are vicinal to a phenolic carbon. The substitution pattern of ring A with a methyl group (Me-8) at C-3 (d 139.4), and a free carboxylic acid (C-7, d 171.5) at C-1 (d 131.9) was confirmed by the HMBC spectrum. Me-8 exhibited correlations to C-2 and C-4, and H-2 correlated with C-7 ( Figure S2). The HMBC correlations between Me-7 0 and C-1 0 confirmed the location of the methoxy group (d 3.32, d 56.0) at C-1 0 (d 165.0). The two aromatic rings were bridged by the carbonyl group C-9 (d 201.7), which was confirmed by HMBC correlations between H-4 (ring A)/H-2 0 (ring B) and C-9. Compound 1 was confirmed as a new sulochrin named 7-demethyl-neosulochrin (1).
Sulochrins 2a, 2 b and S1 are seco-anthraquinones, biosynthesized by ring cleavage of emodin with a questin oxygenase (Fujii et al. 1988). Interestingly, previous evidence showed only one type of sulochrins obtained by cleavage of the C-4a/C-10 bond in emodin, resulting in a product with an acid group formed in the dihydroxylated ring B of the product (Huang et al. 1995). Here we discovered 1 as a new isomer arising from ring cleavage between C-10/ C-10a at the other side of the anthraquinone, forming the carboxylic group at ring A. The proposed biosynthesis of the different sulochrins is shown in Figure S4.
The relative stereochemistry of 3 was deduced by NOESY experiment (Figure S2), where correlations were observed for CH 3 -18 (d 1.30) and H-9 (d 5.57) and between H-5 and Me-25. This confirmed the acetyl residue at C-9 and H-5 in b, and Me-18/H-10 in a-positions. The bridgehead protons H-5/H-10 are in a trans-orientation, thus resulting in the same relative configuration as published for fumigaclavine C (5).
Comparison of the experimental ECD data with the DFT-calculated theoretical spectrum indicated that formula 3 is reflecting also the absolute (5 R,8R,9S,10R) configuration ( Figure S3). Surprisingly, the ECD spectrum of 4 looks similar as that of the 3enantiomer. It should be annotated that the C-8-epimers of 3 can be distinguished sufficiently by DFT calculation of the 13 C NMR data (Table S5).
Fumigaclavines are derived from tryptophan through steps of prenylation, oxygenation, dihydroxylation (Han et al. 2020) and indole ring cleavage (Mutti 2012). The 2,3double bond of indoles undergoes easily an oxidative cleavage, whereby with peracids or dioxygenases e.g., tryptophan is transformed into formylkynurenine and follow-up products (Hirata et al. 1974). Here we hypothesize a potential biosynthetic pathway for fumigaclavines which can be seen in Figure S5. After a C-prenylation on tryptophan, an oxidation occurred at the methyl group to form an aldehyde. An oxidative cyclization led to the bonds formation and decarboxylation. Further oxidation and Nmethylation led to the formation of fumigaclavine B, which is the intermediate for fumigaclavines A, C (5) and I (3). Fumigaclavine A can be formed by acetylation of fumigaclavine B. The structural relation with the fumigaclavines in mind, 3 seems to be formed in such an oxidative cleavage way, so that it is formally a 2,3-seco-derivative of fumigaclavine C (5). For 4, however, we could not see a similar plausible biosynthetic pathway. The NMR shifts previously published for fumigaclavine I (4) are agreeing with our data within the experimental error range, but do not fit on the 13 C NMR shifts calculated for this structure (Table S5). According tothe original report (Shen et al. 2015), the NMR data revealed a mistake in the positioning of the carbonyl group, since a clear doublet of doublet signal was observed for the carbonyl-adjacent methylene (d 3.19, dd, 16.8, 4.2; d 2.55, 16.8, 13.2). This confirms the direct connectivity between the methylene and H-5. We conclude therefore that both compounds are identical, and the previous structure 4 for fumigaclavine I needs to be revised to 3.
Benzoyltryptophan (6) was isolated as a colorless solid. HRMS data established its molecular formula as C 18 H 16 N 2 O 3 . The 1 H NMR spectrum exhibited signals of nine aromatic protons, one methylene and one methine group. The 2 D NMR correlations established a 1,2-disubstituted aromatic ring and one mono-substituted benzoyl fragment ( Figure S2). Besides the signals from the aromatic rings, the 13 C NMR spectrum revealed the presence of two carbonyl groups (d 176.8, 167.8). Finally, HMBC correlations established structure 6 as N-benzoyl tryptophan ( Figure S2, Table S3), which was obtained previously by synthesis . Here, we are reporting N-benzoyl tryptophan for the first time as a natural product.

General experimental procedures
NMR spectra were recorded on a Bruker AMX 300, a 800 MHz Bruker Avance III HD or Varian Inova 500 spectrometer. The 800 MHz Avance III HD spectrometer was equipped with a 5 mm TCI CryoProbe (Bruker Biospin) using standard pulse sequences. 1 H and 13 C chemical shifts are referenced to solvent signals (CD 3 OD: d H ¼ 3.31 and d C ¼ 49.0 ppm; DMSO-d 6 : d H ¼ 2.49 and d C ¼ 39.5 ppm). Optical rotation: Polarimeter (Perkin-Elmer, model 343). ECD spectra: ECD spectra were recorded on a JASCO J-810 spectrometer equipped with a JASCO ETC-505S/PTC-423S temperature controller. UHPLC-HRMS was performed on an Agilent Infinity 1290 UHPLC system equipped with a diode array detector. UV-vis spectra were recorded from 190 to 640 nm. IR data were acquired on Bruker Alpha FTIR spectrometer using OPUS version 7.2. TLC analysis was performed on silica gel plates (Sil G/UV 254 , 0.20 mm, Macherey-Nagel). A Biotage Isolera One Flash Chromatography system was used for flash chromatography and was performed on silica gel 60 (Merck, 0.04-0.063 mm, 230-400 mesh ASTM). Sephadex LH-20 (Amersham Biosciences, Ltd.) was purchased from Sigma-Aldrich Chemie, Steinheim, Germany. All solvents and chemicals used for HRMS and chromatography were VWR Chemicals LC-MS grade, while for extractions, the solvents were of HPLC grade (VWR Chemicals).

Isolation and characterization of the fungal isolate
The strain FVL2 was obtained from the traditional medicinal plant F. vulgare in Egypt. The strain is deposited at Microbial Chemistry Department, National Research Centre (NRC), Egypt. 18S rRNA gene (accession no. MW281863) was obtained and sequenced, which showed 99% similarity to Aspergillus tubingensis ZfF7.2. A phylogenetic tree was constructed ( Figure S100). Further details regarding the fungus isolation/characterization, DNA isolation and 18S rDNA, are shown in supplementary file.

Cytotoxicity assay
Cytotoxicity against the human cell line HL-60 was evaluated using Alamar Blue (Thermos Scientific, Kansas, USA). The assay (Hamid et al. 2004) was performed in 96 well plates (Costar 3595, Corning, New York, USA), with an assay volume of 200 mL. The software Prism 5.03 was used for data analysis (GraphPad Software, USA).

Ab initio calculations
DFT calculations of NMR, OR, and ECD data were performed as described previously, however, SPARTAN'20 was used now, applying the automatic NMR routine (Shaaban et al. 2019).

Conclusions
Three new natural products, 7-demethyl-neosulochrin (1), fumigaclavine I (3) and Nbenzoyl-tryptophan (6) were isolated from a fennel fungal endophyte. The absolute configuration of fumigaclavine I (3) was determined by CD spectra and DFT calculations. The discovery of the new seco-anthraquinone 7-demethyl-neosulochrin (1) revealed a second type of ring cleavage by a questin oxygenase. Our work has broadened the chemistry of Aspergillus as an important source of metabolites.

Disclosure statement
No potential conflict of interest was reported by the authors.