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Abstract

An amino acid-directed strategy has been developed to explore the potential of marine fungi to produce alkaloids. The marine fungus Lecanicillium fusisporum was cultured in glucose-peptone-yeast (GPY) medium to which were added L-tryptophan, L-phenylalanine, L-threonine, D, L-methionine, L-serine, L-lysine and L-valine. A new indole alkaloid, lecasporinoid (1), along with five known alkaloids (2−6) were discovered from the culture broth. The planar structure of lecasporinoid (1) was determined by HR-ESIMS, and 1D and 2D NMR spectroscopic data. The absolute configuration was established by optical rotation and ¹³C NMR calculations combining with a chemical synthetic approach.

Keywords

marine fungus alkaloids amino acid-directed strategy quantum chemical calculations

Introduction

Marine-derived fungi have become an important source of structurally novel and pharmacologically active metabolites. Among them, alkaloids are a class of marine fungal natural products that show unique promise in the development of new drug leads. Biogenetically, alkaloids are derived from an amino acid pathway.¹ Thus, an amino acid-directed strategy, that is adding various natural amino acids, such as L-phenylalanine, L-tryptophan, and L-valine into the regular culture medium, may induce the marine-derived fungi to produce alkaloids. For example, 23 indole alkaloids were obtained from the culture of the marine-derived fungus Fusarium sp. in glucose-peptone-yeast (GPY) medium supplied with L-tryptophan.² Among them, fusaindoterpene B, JBIR-03 and 1,2-bis(1H-indol-3-yl)ethane-1,2-dione showed inhibitory activity against the Zika virus (ZIKV).² Thirteen indole alkaloids, including two new compounds, were discovered from the marine-derived fungus Pseudallescheria boydii F44-1 in GPY medium with L-tryptophan, L-phenylalanine, L-methionine, and L-threonine.³ 3,3’-Cyclohexylidenebis(1H-indole) showed cytotoxic activity against various cancer cell lines.³ Seventeen new fumiquinazoline alkaloids were isolated from the marine fungus Scedosporium apiospermum F41-1 by feeding various amino acids.^4,5 Scedapin C and scequinadoline D displayed significant antiviral activity against hepatitis C.⁴ Additionally, scequinadolines D, E, J and quinadoline A were found to strongly promote triglyceride accumulation in 3T3-L1 cells. Furthermore, qRT-PCR analysis suggested that scequinadoline D might be a potent antidiabetic agent by activating the PPARγ pathway.⁵ These results demonstrate the great potential of alkaloid production in marine fungi.

Recently, a marine fungus Lecanicillium fusisporum was isolated from the inner tissue of a sea cucumber collected from Xisha Islands in China. To induce this fungal strain to produce alkaloids, L. fusisporum was fermented in GPY medium supplemented with L-tryptophan, L-phenylalanine, L-threonine, D, L-methionine, L-serine, L-lysine and L-valine. By tracking the aromatic ¹H NMR signals at δ_H 6.50 − 8.50 in the EtOAc extract of the culture broth, one new indole alkaloid, lecasporinoid (1), together with five known alkaloids (2–6) were discovered (Figure 1). Herein, we report the isolation, structure elucidation and bioactivity evaluation of these compounds.

Figure 1.

Chemical formulas of compounds 1 to 6.

Results and Discussion

Structural Elucidation

Lecasporinoid (1) was obtained as a colorless oil. The molecular formula was established as C₁₆H₁₉NO₄ based on the potassium molecular ion detected at m/z 328.0954 (calcd 328.0946 for C₁₆H₁₉NO₄K) in the HR-ESIMS. The ¹³C NMR spectrum (Table 1), in combination with DEPT and HSQC spectra, displayed 16 carbon signals, consisting of three methyls, one sp³ methylene, five sp² and two oxygen atom bearing methines, three nonprotonated sp² carbons and two carbonyl carbons. The ¹H-¹H COSY correlations of H-4/H-5, H-5/H-6, H-6/H-7, as well as the HMBC correlations (Figure 2) from H-2 to C-3/C-4a/C-7a, from H-4 to C-7a, from H-7 to C-4a, and from H₂-8 to C-3/C-4a/C-9, suggested that the core structure of 1 is indole-3-acetic acid. Moreover the ¹H-¹H COSY correlations of Me-14/H-10, H-10/H-11, H-11/Me-15 and the HMBC correlations from H-11 to C-12, from Me-13 to C-12, combining with the deshielded signals of C-11 (δ_C 71.4) and H-11 (δ_H 5.03), displayed the presence of the substructure −CH(CH₃)−CH(CH₃)−O−C(=O)−CH₃. Then, C-8 was connected to C-10 by an ester group, which was supported by HMBC correlations from H₂-8 and H-10 to C-9. Accordingly, the planar structure of 1 was established, as shown in Figure 1.

Figure 2.

Key ¹H–¹H COSY and HMBC correlations of compound 1.

Table 1.

¹H NMR (500 MHz) and ¹³C NMR (125 MHz) Data for 1, 1a and 7 in CDCl₃ (J in Hz).

Position	1		1a		7
Position	δ_C, type	δ_H, mult. (J in Hz)	δ_C, type	δ_H, mult. (J in Hz)	δ_C, type	δ_H, mult. (J in Hz)
1-NH		8.09, brs		8.29, brs		8.09, brs
2	123.1, CH	7.18, m	123.3, CH	7.15, m	123.1, CH	7.19, m
3	108.7, C		108.3, C		108.8, C
4a	127.4, C		127.2, C		127.3, C
4	119.1, CH	7.62, brd (8.0)	118.9, CH	7.64, brd (8.0)	118.8, CH	7.63, brd, (8.0)
5	119.8, CH	7.13, brt (8.0)	119.7, CH	7.12, brt (8.0)	120.0, CH	7.15, brt (8.0)
6	122.4, CH	7.19, brt (8.0)	122.2, CH	7.20, brt (8.0)	122.6, CH	7.21, brt (8.0)
7	111.3, CH	7.37, brd (8.0)	111.3, CH	7.32, brd (8.0)	111.5, CH	7.37, brd (8.0)
7a	136.2, C		136.2, C		136.3, C
8	31.6, CH₂	3.78, s	31.7, CH₂	3.78, s	31.9, CH₂	3.83, d (15.5) 3.78, d (15.5)
9	171.5, CO		171.6, CO		171.8, CO
10	71.7, CH	5.03, dq (6.5, 3.5)	71.9, CH	5.01, m	75.6, CH	4.73, m
11	71.4, CH	4.97, dq (6.5, 3.5)	71.6, CH	4.96, m	70.2, CH	3.68, m
12	170.6, CO		170.6, CO		16.5, CH₃	1.22, d (6.5)
13	21.2, CH₃	1.95, s	20.9, CH₃	1.86, s	19.0, CH₃	1.08, d (6.5)
14	15.0, CH₃	1.21, d (6.5)	16.1, CH₃	1.22, d (6.5)
15	15.2, CH₃	1.16, d (6.5)	16.2, CH₃	1.16, d (6.5)

To determine the absolute configuration of 1, quantum chemical calculations were made using Gaussian 09 software. By comparing the experimental optical rotation value (+23) with the calculated value of all configurations (10R,11R-1, −53.6; 10R,11S-1, −47.5; 10S,11R-1, +52.1; 10S,11S-1, +52.3), the chirality at C-10 was deduced to be 10S. Then, the correlation coefficient (10S,11R-1: 0.99909; 10S,11S-1: 0.99877) and DP4+ probability (10S,11R-1: 98.43; 10S,11S-1: 1.57) between the calculated ¹³C NMR data (Figure 3) and experimental data suggested that 10S,11R-1 was a better match.

Figure 3.

¹³C NMR calculation results for two possible isomers of 1. (a) Linear correlation plots of calculated versus experimental ¹³C NMR chemical shift values for each potential configuration. (b) Relative errors between the predicted ¹³C NMR chemical shifts of two potential structures and recorded ¹³C NMR data. (c) The DP4+ probability of chemical shifts about isomer 1 (10S,11S-1) and isomer 2 (10S,11R-1).

Finally, in order to validate further the calculation, we turned our attention to synthesize (10R,11R)-lecasporinoid (1a) for comparison. Synthesis of 1a was performed in two sequential steps, as illustrated in Figure 4. First, commercially available indole-3-acetic acid activated with 1-ethyl-3(3-dimethylpropylamine) carbodiimide hydrochloride (EDCI) in dichloromethane (DCM) was treated with (2R,3R)-butanediol to obtain 7.⁶ In the subsequent step, the acetylation of compound 7 with acetic anhydride was carried out in the presence of N,N-diisopropylethylamine (DIPEA). The resulting 1a was purified from the reaction mixture by silica gel chromatography in good yield. Its spectroscopic data (¹H and ¹³C NMR) differ from those of lecasporinoid (1), excluding the possibility of 10S,11S-1. Thus, the absolute configuration was assigned as 10S,11R-1.

Figure 4.

The synthetic route of 1a. Reagents and conditions: (a) (2R,3R)-butanediol, EDCI, DMAP, DCM, rt, 12 h; (b) acetic anhydride, DMAP, DIPEA, DCM, rt, 1.5 h.

1,1,1-Tris(3-indolyl)methane (2),⁷ 3-hydroxy-β-carboline (3),⁸ 3,6-bis(1-methylethyl)-2(1H)-pyrazinone (4),⁹ deoxymutaaspergillic acid (5)¹⁰ and 6-[(1S)-1-methylpropyl]-3-(2-methylpropyl)-2(1H)-pyrazinone (6)¹⁰ were identified by comparing their ¹H and ¹³C data with those in the literature.

Anti-DENV Activity

To investigate the antidengue virus 2 activity of compounds 1 to 6 from L. fusisporum, in the DENV2 16681 replication assay, A549 cells were treated with compounds 1 to 6 at the indicated concentrations for 3 days. Then the effects of compounds 1 to 6 on DENV2 replication were analyzed by standard focus-forming assay. However, the assay revealed that compounds 1 to 6 have no apparent effect on dengue virus.

Experimental

General Experimental Procedures

1D and 2D NMR spectra were measured in CDCl₃ using Bruker Avance II 400 and 500 spectrometers (Bruker Bio Spin AG, Industriestrasse 26, Fällanden, Switzerland), and the chemical shifts are given relative to the residual solvent signals (CDCl₃: δ_H 7.26, δ_C 77.2). UV spectra were recorded using a Shimadzu UV-Vis-NIR spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan), optical rotations (OR) were measured with an Anton Paar MCP500 polarimeter at 25 °C, and IR spectra were recorded on a Bruker tensor-27 spectrophotometer with KBr discs. Mass spectra were acquired on Thermo DSQ EI low-resolution and the Thermo MAT95XP EI high-resolution mass spectrometers (Thermo Fisher Scientific Inc., Waltham, MA, USA). Preparative HPLC was performed using a Shimadzu LC-20AT HPLC pump (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) installed with an SPD-20A dual λ absorbance detector (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) and a Shim-pack PRC-ODS HPLC column (250 × 20 mm, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan). Column chromatography was performed over silica gel (200 − 300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (Green Herbs, Beijing, China).

Fungal Strain and Culture Method

The marine fungus Lecanicillium fusisporum was isolated from the inner tissue of a sea cucumber collected from Xisha Islands in China. The fungal strain was maintained on potato dextrose agar (PDA) slants. Analysis of the ITS rDNA by BLAST database screening provided a 99.9% match with Lecanicillium fusisporum. The fermentation medium contained glucose 10 g/L, peptone 5 g/L, yeast extract 2 g/L, sea salt 20 g/L, L-tryptophan 2 g/L, L-phenylalanine 2 g/L, L-threonine 2 g/L, D, L-methionine 2 g/L, L-serine 2 g/L, L-lysine 2 g/L, L-valine 2 g/L, and H₂O 1 L (pH 7.5). The mycelia were aseptically transferred to 500 mL Erlenmeyer flasks containing 200 mL of the liquid medium, then sterilized at 120 °C for 30 min. The flasks were statically incubated at 28 °C for 40 days.

Extraction and Isolation

Fifty liters of culture broth were filtered through cheesecloth. The culture broth was extracted three times with 60 L EtOAc. The combined EtOAc extract was concentrated under reduced pressure to afford 17 g crude extract. This was chromatographed on a silica gel column (diameter: 8 cm, length: 80 cm, silica gel: 170 g) with a stepwise gradient of light petroleum–EtOAc (100:0–0:100), followed by EtOAc–MeOH (100:0–0:100). The eluents were collected every 400 mL, and similar fractions were pooled to afford six fractions (code Fr.1–Fr.6) by TLC monitoring. Fr.3 was further chromatographed using Sephadex LH-20 (MeOH) to give three subfractions (Fr.3-1–Fr.3-3). Fr.3-2 was further purified by preparative HPLC (73% MeOH–H₂O) to yield compounds 4 (2.2 mg) and 5 (3.2 mg). Fr.4 was subjected to silica gel column chromatography eluting with a gradient of light petroleum–EtOAc to afford five subfractions (Fr.4-1–Fr.4-5). Fr.4-3 was further purified by preparative HPLC (71% MeOH–H₂O) to yield compounds 1 (0.5 mg), 2 (1.8 mg) and 3 (1.0 mg). Fr.4-4 was further purified by preparative HPLC (68% MeOH–H₂O) to obtain compound 6 (2.0 mg).

Procedure for the Preparation of Compounds 7 and 1a

(2R,3R)-3-Hydroxybutan-2-yl-2-(1H-indol-3-yl)acetate (7). To a solution of 3-indoleacetic acid (350 mg, 2 mmol) and (2R,3R)-butanediol in dry dichloromethane (10 mL) at room temperature was added EDCI (383 mg, 2 mmol) and DMAP (cat.). After 12 h, the reaction mixture was quenched with water (10 mL) and extracted with dichloromethane (3 × 8 mL). The combined organic phases were dried with anhydrous sodium sulfate and evaporated to dryness. Purification by column chromatography afforded 7 (236 mg, 48%) as a white solid. ¹H NMR and ¹³C NMR data see Table 1.

(2R,3R)-Lecasporinoid (1a). To a solution of 7 (94 mg, 0.38 mmol) and acetic anhydride (77 mg, 0.76 mmol) in dry dichloromethane (6 mL) at room temperature was added DIPEA (54 mg, 0.42 mmol) and DMAP (4.6 mg, 0.038 mmol). After 1.5 h, the reaction mixture was quenched with water (8 mL) and extracted with dichloromethane (3 × 6 mL). The combined organic phases were dried with anhydrous sodium sulfate and evaporated to dryness. Purification by column chromatography afforded 1a (99 mg, 90%) as a colorless oil. ¹H NMR and ¹³C NMR data see Table 1.

Spectroscopic Data

Lecasporinoid (1): colorless oil; [α]20 D +23.0 (c 1.0, MeOH); UV (MeOH) λ_max (log ε) 280 (2.21) nm; IR (KBr) ν_max 3401, 2976, 1087, 1049, 1024, 1002 cm⁻¹; ¹H NMR and ¹³C NMR data see Table 1; HRESIMS m/z 328.0954 [M + K]⁺ (calcd for C₁₆H₁₉NO₄K, 328.0946).

Antiviral Activity Against DENV2

A549 cells were pretreated with compounds 1 to 6 (5 μM or 50 μM) for 1 h, followed by infection with DENV2 16681 strains at MOI 3. The cells were incubated with the chemicals throughout the entire experiment. Supernatants were harvested at 24 h p.i. for virus titration by standard focus-forming assay (FFA) on Vero cells. Samples were diluted in DMEM supplemented with 2% FBS. Vero cells were inoculated with serial 10-fold dilutions of each virus sample. Overlay containing 1% methylcellulose (Sigma) and 2 × DMEM (Invitrogen) was then added and incubated at 37 °C. Cells were washed with phosphate-buffered saline (PBS) and fixed with 1% paraformaldehyde (PFA) in PBS at 3 days p.i. Cells were incubated with anti-WNV E18 monoclonal antibody (MAb), followed by incubation with goat anti-mouse HRP-conjugated secondary antibody (CST). Cells were incubated with TrueBlue peroxidase substrate (KPL). The number of spots was determined by a CTL-BioSpot analyzer.

Computational Section

The absolute configuration of compound 1 was determined by quantum chemical calculations using Gaussian 09 software. Conformational analysis of diastereoisomers was performed by using the Merck Molecular Force Field (MMFF).¹¹ They were further optimized by the density functional theory (DFT) method at the B3LYP/6-31G(d) level. The optical rotations were calculated using the TDDFT method at the B3LYP/6-311 + G(d) level in methanol (λ = 589.3 nm). The ¹³C NMR were calculated using the TDDFT at the B3LYP /6-31G* (d, p) level in chloroform. The predicted chemical shifts and optical rotations were obtained according to the Boltzmann distribution theory and their relative Gibbs free energy. All the theoretical NMR values were analyzed by using linear regression and DP4+.

Ethical Approval

Ethical Approval is not applicable for this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

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There are no human subjects in this article and informed consent is not applicable.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211046072 - Supplemental material for Alkaloids From the Marine Fungus Lecanicillium fusisporum Using an Amino Acid-Directed Strategy

Supplemental material, sj-docx-1-npx-10.1177_1934578X211046072 for Alkaloids From the Marine Fungus Lecanicillium fusisporum Using an Amino Acid-Directed Strategy by Qin Huang, Hou-Jin Li, Chang-Bai Huang, Zi-Han Wang, Wen-Jian Lan and Lai-You Wang in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Guangdong Provincial Science and Technology Research Program (grant number Nos. 2015A020216007, Nos. 2016A020222004), and the National Science Foundation of China (grant number No. 81673689, No. 81872795).

Trial Registration

Not applicable, because this article does not contain any clinical trials.

ORCID iD

Wen-Jian Lan

Supplemental Material

Supplemental material for this article is available online.

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