Exploration of Indole Alkaloids from Marine Fungus Pseudallescheria boydii F44-1 Using an Amino Acid-Directed Strategy

The composition of the culture medium has great influence on the metabolite production of the marine fungus Pseudallescheria boydii F44-1. By adding amino acids to GPY culture medium, two new bisindole alkaloids, pseudboindoles A and B (1 and 2), together with 11 known indole alkaloids were isolated from the culture broth. Their structures were elucidated by comprehensive analysis of the NMR, MS, IR, and UV spectra. The 3,3′-cyclohexylidenebis(1H-indole) (3) showed cytotoxic activity against various cancer cell lines.


Introduction
Marine indole alkaloids are an increasingly growing class of secondary metabolites. From 2003 to 2015, about 800 new marine indole alkaloids were obtained [1]. Indole alkaloids-including mono-, bisand trisindole alkaloids-are biosynthetically derived from one-, two-and three-indole building blocks, respectively, and have high structural diversity. Some of them were found to possess diverse biological activity, such as cytotoxic, antiviral, antiplasmodial, antifungal, antibacterial and anti-inflammatory activity, and are therefore promising leads for new drug development [2][3][4][5]. The natural occurrence of indole alkaloids is the result of biosynthesis via the coupling of the inessential amino acid tryptophan with other amino acids and structural fragments. Based on this consideration, our research group established an amino acid-directed strategy to explore the potential of marine fungi to produce diverse alkaloids. To date, more than forty novel and/or bioactive indole alkaloids have been obtained from marine fungi. For example, when cultured in glucose-peptone-yeast (GPY) extract medium supplemented with L-tryptophan, L-phenylalanine, L-threonine, and D,L-methionine, the marine fungus Scedosporium apiospermum F41-1 produced 12 new indole alkaloids. Among them, scedapin C and scequinadoline D displayed significant antiviral activity against hepatitis C [6]. A total of 18 indole alkaloids were isolated from the marine fungus Dichotomomyces cejpii F31-1 by feeding it with L-tryptophan and L-phenylalanine [7]. Scequinadoline A possesses the potential for further development as a dengue virus inhibitor [8].
Recently, another marine fungus Pseudallescheria boydii (collection no. F44-1) was isolated from the soft coral Sarcophyton sp. collected in the Hainan Sanya National Coral Reef Reserve, China. This fungal strain was cultured in GPY medium and GPY medium supplied with amino acids, including L-tryptophan, L-phenylalanine, L-methionine, and L-threonine. The culture extracts were analyzed with HPLC detected at UV 254 nm. HPLC traces indicated that Pseudallescheria boydii F44-1 cultured in the GPY medium containing additional amino acids could produce more metabolites with strong UV absorption ( Figure 1 and Supplementary Figure S1). This meant that amino acids could regulate the production of metabolites containing aromatic rings. By tracking the characteristic 1 H NMR signals in the aromatic region 6.5−8.5 ppm, two new bisindole alkaloids pseudboindoles A and B (1 and 2), together with 11 known indole alkaloids ( Figure 2), were obtained efficiently. Here, we reported the isolation, structure elucidation and cytotoxic activity of these compounds.

Fungal Strain and Culture Method
The marine fugus Pseudallescheria boydii (collection no. F44-1) was isolated from the inner tissue of the soft coral Sarcophyton sp. collected from Hainan Sanya National Coral Reef Reserve, China. This fungal strain was conserved in 15% (v/v) glycerol aqueous solution at −80 • C. A voucher specimen was deposited in the School of Chemistry, Sun Yat-sen University, Guangzhou, China. Analysis of the ITS rDNA by BLAST database screening provided 99.9% match to Pseudallescheria boydii.
The fermentation medium was glucose 15 g, peptone 10 g, yeast extract 2 g, L-tryptophan 2 g, L-phenylalanine 2 g, L-methionine 2 g, L-threonine 2 g, sea salt 25 g, and H 2 O 1L at pH 7.5. Fungal mycelia were cut and transferred aseptically to 1000 mL conical flasks each containing 400 mL sterilized liquid medium. The flasks were incubated at 28 • C for 20 days.

Extraction and Isolation
A total of 60 liters of liquid culture were filtered through cheesecloth. The culture broth was successively extracted five times with EtOAc (60 L). Finally, the extract was concentrated by low-temperature rotary evaporation to obtain a crude extract (39.8 g).

Cytotoxicity Assay
The in vitro cytotoxic activity of 1−13 was determined by means of the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The tested human cancer cell lines were seeded in 96-well plates at a density of 3 × 10 7 cells/L, and the compounds were added at various concentrations (7.864−30.00 µM). After 72 h, MTT was added to the culture medium at a final concentration of 0.5 mg/mL, and the plates were incubated for 4 h at 37 • C. The supernatant was removed. The formazan crystals were dissolved in DMSO (150 µL) with gentle shaking at room temperature. The absorbance at 570 nm was recorded with a microplate reader (Bio-Rad, Hercules, CA, USA), and the data were analyzed with the SPSS (version 13.0) [25].

Conclusions
By tracking characteristic 1 H NMR signals in the aromatic region of 6.50−8.50 ppm, two new bisindole compounds, pseudboindoles A and B (1 and 2), together with 11 known indole alkaloids (3−12) were efficiently isolated from the marine fungus Pseudallescheria boydii F44-1. The 3,3 -cyclohexylidenebis(1H-indole) (3) showed significant cytotoxic activity against various cancer cell lines. The result proves again that an amino acid-directed strategy is effective for inducing the marine fungi to produce diverse alkaloids. However, the specific quantitative relationship between amino acids and alkaloids and their biosynthesis pathways still need further study. After revealing these relationships, the application of this strategy will be more efficient.