Fusaripyridines A and B; Highly Oxygenated Antimicrobial Alkaloid Dimers Featuring an Unprecedented 1,4-Bis(2-hydroxy-1,2-dihydropyridin-2-yl)butane-2,3-dione Core from the Marine Fungus Fusarium sp. LY019

The fungal strain, Fusarium sp. LY019, was obtained from the Red Sea sponge Suberea mollis. Bioassay-directed partition of the antimicrobial fraction of the extract of the culture of the fungus provided two dimeric alkaloids, fusaripyridines A and B (1 and 2). The compounds possess a previously unreported moiety, 1,4-bis(2-hydroxy-1,2-dihydropyridin-2-yl)butane-2,3-dione. Further, the compounds display a highly oxygenated substitution pattern on the dihydropyridine moieties, representing an additional feature of the fusaripyridines. Fusaripyridines A and B are the first examples of natural products possessing 1,4-bis(2-hydroxy-1,2-dihydropyridin-2-yl)butane-2,3-dione backbone. Careful analyses of the one- and two-dimensional NMR and HRESIMS spectra of the compounds secured their structural mapping, while their absolute stereochemistry was established by analyses of their ECD spectra. The production of such dimeric alkaloids with an unprecedented moiety in the culture of Fusarium sp. LY019 supports further understanding of the biosynthetic competences of the cultured marine-derived fungi. Fusaripyridines A and B selectively inhibited the growth of Candida albicans with MIC values down to 8.0 µM, while they are moderately active against S. aureus, E. coli and HeLa cells.


Introduction
Investigation of fungi of marine origin has been mostly ignored for several years as a result of their low occurrence and the doubt about their true existence. Recently, this situation has been changed due to the appreciation that marine-derived fungi embody a relatively diverse class and represent an enormous source of bioactive secondary metabolites. It was found that altering the culture conditions of the fungi, such as addition of sodium chloride for example, stimulates the production of new secondary metabolites not sodium chloride for example, stimulates the production of new secondary metabolites not reported under regular culture conditions [1][2][3]. Several cultivation-dependent investigations showed that marine algae and sponges, for example, represent a massive source for fungi [2,[4][5][6][7]. Moreover, an unpredicted high fungal variety was found in deep-sea hydrothermal ecosystems using molecular approaches [8]. Recently, a growing number of new fungal secondary metabolites with promising pharmaceutical bioactivities has been discovered, representing potential candidates for drug development. The biotechnological potential of the endophytic fungi has been demonstrated in different applications of the life science as antiviral agents, antifungal agents, antibacterial agents, anticancer drugs and in the biological control of different contaminants and plague [9,10]. Such biological properties are attributed to the unique and diverse fungal secondary metabolites with biotechnological applications and pharmaceutical importance [10,11].
Members of the genus Fusarium are widely spread in soils and plants worldwide and represent a central source of plant pathogens in agriculture [12,13]. They are also considered as a rich source of bioactive compounds. Representative examples of Fusarium-derived natural products include fusarins A, C, D, and F [14] and lucilactaene [15]. Lucilactaene, a metabolite of the Fusarium species, has received widespread attention in terms of both biosynthesis investigation and total chemical synthesis [16,17]. Lucilactaene is considered as inhibitor of cell cycle in p53-transfected cancer cells [15].

Purification of Fusaripyridines A and B (1 and 2)
The marine-derived fungus Fusarium sp. LY019 (Figure 1) was obtained from the inner tissue of the sponge Suberea mollis (Figure 1). Chromatographic purification of the antimicrobial fraction of the extract of the fermentation broth afforded two alkaloids, fusaripyridines A and B (1 and 2) ( Figure 2).

Structure of Fusaripyridine B (2)
Fusaripyridine B (2) (Figure 2), an optically active compound, possesses a molecular formula C18H24N2O12 as supported by HRESIMS ( Figure S8), being 32 mass unit large than 1, suggesting additional two oxygen atoms in the molecule. Its 13 C and 1 H NMR spectra (Table 1) Figure S14) experiments confirmed its planar structure. Therefore, the oxygen atoms accounted for two additional hydroxy groups located at N-1 and N-1', the sole positions allowed for substitution in 2.

Biological Evaluation of the Compounds
The fusaripyridines A (1) and B (2) are evaluated, in a disc diffusion assay, for their antimicrobial effects against C. albicans, E. coli and S. aureus at a concentration of 50 µg/disc. The compounds displayed potent antifungal effects against C. albicans with inhibition zones of 26 and 24 mm, and with MIC values of 8.0 and 8.0 µM, respectively. Further, the compounds displayed weak growth inhibition towards S. aureus and E. coli with MIC of ≥32.0 µM (Table 2). Moreover, fusaripyridines A (1) and B (2) showed weak effects

Structure of Fusaripyridine B (2)
Fusaripyridine B (2) (Figure 2), an optically active compound, possesses a molecular formula C 18 H 24 N 2 O 12 as supported by HRESIMS ( Figure S8), being 32 mass unit large than 1, suggesting additional two oxygen atoms in the molecule. Its 13 C and 1 H NMR spectra (Table 1) Figure S14) experiments confirmed its planar structure. Therefore, the oxygen atoms accounted for two additional hydroxy groups located at N-1 and N-1', the sole positions allowed for substitution in 2.

Biological Evaluation of the Compounds
The fusaripyridines A (1) and B (2) are evaluated, in a disc diffusion assay, for their antimicrobial effects against C. albicans, E. coli and S. aureus at a concentration of 50 µg/disc. The compounds displayed potent antifungal effects against C. albicans with inhibition zones of 26 and 24 mm, and with MIC values of 8.0 and 8.0 µM, respectively. Further, the compounds displayed weak growth inhibition towards S. aureus and E. coli with MIC of ≥32.0 µM (Table 2). Moreover, fusaripyridines A (1) and B (2) showed weak effects towards HeLa cells with IC 50 of ≥25 µM. The above data suggest the selectivity of the C. albicans towards fusaripyridines A and B.

General Experimental Procedures
Optical rotations values were measured on a digital DIP-370 polarimeter (JASCO, Oklahoma City, OK, USA), while the UV spectra are obtained on a Hitachi 300 spectrometer (Hitachi High-Technologies Corporation, Kyoto, Japan). The ECD spectra were obtained on a JASCO J-810 spectropolarimeter (JASCO, Oklahoma City, OK, USA) with a 0.5 cm cell in MeOH. The NMR spectra were acquired on Bruker Avance DRX 600 MHz spectrometer (Bruker, Rheinstetten, Germany). Positive ion HRESIMS spectra were obtained on a Thermo LTQ Orbitrap XL mass spectrometer (Thermo Finnigan, Bremen, Germany). Normal SiO 2 (Merck, Darmstadt, Germany) and Sephadex LH 20 (Pharmacia, Uppsala, Sweden) were used for chromatography.

Host Organism, Suberea mollis
The Red Sea sponge Suberea mollis (Figure 1) was harvested by hand using scuba from Yanbu at the Saudi Red Sea at a depth of −30 m. Description of the sponge was reported earlier in details [32].

Preparation of the Fungal Isolate LY019
The fungal isolate Fusarium sp. LY019 was isolated from the Red Sea sponge Suberea mollis by culturing on Czapek-Dox yeast agar medium (K 2 HPO 4 0.1 g, MgSO 4 ·7H 2 O 0.5 g, NaNO 3 3.0 g, KCl 0.5 g, FeSO 4 0.01 g, sucrose 30.0 g, agar 20.0 g, pH 6.7), using several purification steps until a pure isolate was obtained. After that, the isolate was inoculated into 50 mL of Czapek-Dox broth in 200 mL flasks at pH 7 and was incubated at 160 rpm, 25 • C for 7 days.

Preparation of Genomic DNA of Isolate LY019
Subculture of the isolate in corresponding broth for 4 days at 25 • C was carried out. Afterwards the mycelia were separated using a vacuum filtration unit. The mycelial mat was dried and powdered. The fungal DNA was obtained using QIAamp DNA Mini Kit (Qiagen, Hilden, Duesseldorf, Germany) as stated by the manufacturer.

Amplification of ITS-rDNA Fragments of Isolate LY019
Using ITS1 and ITS4 primers, the genomic DNA was served as the template for amplification of the fungal ITS-rDNA fragments [33]. The PCR reaction mixture for amplification and the reaction conditions are similar with previous work [22]. The agarose Gel DNA purification kit (Qiagen, Hilden, Duesseldorf, Germany) was used for purification of the PCR products.

Sequence of ITS-rDNA Regions of Isolate LY019
The sequence of the ITS-rDNA regions was compared with correlated sequences in NCBI [34]. Editing and alignment of the ITS-rDNA sequence with the best n-BLAST hits in GenBank were obtained using the Clustal X (version 1.83) program (Conway Institute, University College Dublin) [35]. The adjustment was achieved using BioEdit software (Bioz, Inc., Los Altos, Ca 94022, USA) manually [36]. MEGA 5 program was used for the base composition of the sequence [37].

Characterization of the Fungal Isolate LY019
The resulted sequence analysis exhibited 99% identity with Fusarium commune (NCIB accession number KU891512).

Fermentation and Extraction of the Broth
The fermentation was obtained in a 2.8 L Fernbach flasks with one liter of Czapek-Dox medium (Difco) containing 3% NaCl (w/v). Each flask was inoculated with 10 mL of the seed followed by incubation at 27 • C and 180 rpm for 10 days. After that, each flask was mixed with ethyl acetate (500 mL × 2) and shaken at 1800 rpm for 30 min. The organic phase was separated and dried under vacuum.

Computational Details
Calculations of the DFT are performed using Gaussian 16 W [38]. A conformation analysis using the GMMX plugin followed by a geometry optimization at the B3LYP/6-31g(d) level was conducted. Similarly, a frequency check at the same level of theory was performed. In addition, a 0.5 kcal.mol −1 cutoff was used to select only the most stable conformer. Rotational strengths were calculated on 20 excited states using the b3lyp/6-31g(d) level of theory. Finally, Gaussview 6 was used to plot the ECD spectra. The XYZ coordinates of the most stable conformer was added in the supporting data (Supporting data s2).

Antimicrobial Activities of the Compounds
The antimicrobial effects of 1 and 2 against C. albicans, E. coli and S. aureus were carried out at 50 µg/disc as previously reported in a disk diffusion assay [39][40][41][42].

Evaluation of the MIC
Determination of the MIC of 1 and 2 against C. albicans, E. coli and S. aureus was performed in a macrodilution assay as described earlier [40,43].

Conclusions
The organic extract of the fungus Fusarium sp. LY019 yielded two alkaloids, fusaripyridines A (1) and B (2), with an unprecedented 1,4-bis(2-hydroxy-1,2-dihydropyridin-2-yl)butane-2,3-dione backbone. Analyses of the spectroscopic data (NMR and HRESIMS) supported the planar structures of 1 and 2, while the absolute configurations were obtained from the comparison of their ECD spectra. Fusaripyridines A and B are highly oxygenated alkaloids on the dihydropyridine moieties and represent the first candidates in this class. The compounds displayed selective and potent activities towards C. albicans with MIC values of 8.0 and 8.0 µM. The current results highlight the biosynthetic competences of fungi of marine origin as a foundation of drug leads with pharmaceutical potential. Fusaripyridines A and B could serve as a model for the discovery of novel antibiotics.