New Fusarin Derivatives from the Marine Algicolous Fungus Penicillium steckii SCSIO41040

Five new fusarin derivatives, steckfusarins A–E (1–5), and two known natural products (6, 7), were isolated and identified from the marine algicolous fungus Penicillium steckii SCSIO 41040. The new compounds, including absolute configurations, were determined by spectroscopic analyses and calculated electronic circular dichroism (ECD). All new compounds were evaluated for their antioxidant, antibacterial, antifungal, antiviral, cytotoxic, anti-inflammatory, antioxidant, cholesterol-lowering, acetyl cholinesterase (AChE) enzyme and 6-phosphofructo-2-kinase (PFKFB3) and phosphatidylinositol-3-kinase (PI3K) inhibitory activities. The biological evaluation results revealed that compound 1 exhibited radical scavenging activity against 2,2-diphenyl-1-picrylhydrazylhydrate (DPPH), with an IC50 value of 74.5 µg/mL. In addition, compound 1 also showed weak anti-inflammatory activity at a concentration of 20 µM.


Introduction
Based on genome sequencing and mining and antimicrobial screening of crude extracts, P. steckii has shown strong activity against some pathogenic bacteria [1].It has been recently reported that the secondary metabolites of P. steckii were mainly tanzawaic acids and alkaloids that demonstrated a broad range of significant biological activities, such as antimicrobial, anti-inflammatory and lipid-lowering activities [2][3][4][5][6].
Fusarins are a class of mycotoxins characterized by a 2-pyrrolidone moiety and a triene side chain of twelve carbon atoms [7] that are usually isolated from Penicillium sp.[8].The compounds were biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) [9].In the human neuroblastoma cell line SH-SY5Y, fusarin derivatives were first proven to effectively promote neurite growth and block the cell cycle in the G 0 /G 1 phase [10].Fusarin derivatives inhibit mammalian DNA polymerases and human DNA topoisomerase II in vitro [11].Additionally, they induce the formation of 6-thioguanine-resistant mutants in V79 cells, as well as the asynchronous replication of polyoma DNA sequences subsequent to the presence of a microsomal activation system [12,13].Fusarins have been reported to be unstable after prolonged exposure to ultraviolet light and elevated temperatures [14]; additionally, light can induce the rearrangement of fusarins, leading to the simultaneous appearance of fusarin analogues [15].
During our continuous study exploring new bioactive natural products from marine microorganisms [16,17], five new fusarin derivatives (1-5) (Figures S1-S4) and two known During our continuous study exploring new bioactive natural products from marine microorganisms [16,17], five new fusarin derivatives (1-5) (Figures S1-S4) and two known natural products (6, 7) (Figure 1) were obtained from the fungus Penicillium steckii SCSIO 41040, which was isolated from a green algae Botryocladia sp. of the South China Sea.Herein, we describe the fermentation, isolation, structural determination and biological activities of these compounds.
of the HMBC spectrum, the acetic acid group was confirmed by the HMBC correlations from H3-22 (δH 1.98) to C-26 (δC 170.8) and H2-25 to C-26.The E-configuration of the ∆ 6 (7)  double bond was determined by the coupling constant (J = 15.6 Hz).NMR data analyses of 3 showed a similar multiplicity pattern in the glycerol moiety, indicating that they are two components and have the same plane structure.The resonances of the glycerol moiety suggested them to be diastereoisomers presenting in a ratio of 3:1 (3a:3b), which may be formed by Michael addition [21].NOESY correlations (Figure 4) of 3a indicated the Econfiguration for the other double bonds in the side chain and the cofacial orientation of H-13, H-14, CH3-18 and H-24.The NOESY spectrum of 3b was the same as 3a, except the signal of H-24 was the opposite.These data permitted assignment of the (13R*, 14R*, 15R*, 24R*)-3a and (13R*, 14R*, 15R*, 24S*)-3b relative configurations [15,19].Compound 4 was obtained as a yellow oil.Its HRESIMS ([M + H] + , 346.2016; calcd for C20H28NO4 + , 346.2013) data were in agreement with the molecular formula C20H27NO4.Its 1 H NMR and 13 C NMR data (Table 3) closely resembled the data for 2, except for the absence of the hydroxyl group and an epoxy system at the 2-pyrrolidone ring structure.To data, all fusarin derivatives reported from natural sources have had a free hydroxyl group   13 C NMR data (Table 3) closely resembled the data for 2, except for the absence of the hydroxyl group and an epoxy system at the 2-pyrrolidone ring structure.To data, all fusarin derivatives reported from natural sources have had a free hydroxyl group at C-15; however, compound 4 has no such substituent [22][23][24].The relative configuration of 4 was confirmed by its NOESY correlations and coupling constants (Figure 4).The NOESY correlations of H 3 -1 (δ H 1.64)/H-4 (δ H 5.95), H-4/H-6 (δ H 6.26), H 2 -9 (δ H 2.38)/H 3 -21 (δ H 1.72) and the large coupling constant of H-6/H-7 (J = 15.6 Hz) suggested the E geometry of all double bonds.Each pair of 1 H and 13 C NMR signals showed a similar multiplicity pattern, suggesting that compound 4 was an inseparable mixture of two geometric isomers presenting in a ratio of 1:1 (4a:4b).Even under mild conditions, the stereo center of C-15 was easy to be epimerized through ring opening [19,20].Comparing 4a and 4b (Table 2), the main difference occurred at the configuration at C-13, C-15 and C-18, which was proven by the carbon chemical shifts of C-13 (∆δ C 1.1 ppm), C-15 (∆δ C 1.1 ppm) and C-18 (∆δ C 0.2 ppm).The NOESY spectrum of 4a showed cross-peaks between H-13 (δ H 4.33) and H 3 -18 (δ H 1.11) that indicated H-13 and H-15 (δ H 1.48) were on the opposite side, whereas no relevant signal between H-13 and H 3 -18 was found in 4b.Accordingly, the relative configurations of 4a and 4b could be assigned as (13R*, 15R*)-4a and (13R*, 15S*)-4b.Compound 5 was obtained as a yellow oil.It showed a [M + H] + ion peak at m/z 265.1431 in the positive-ion HRESIMS (calcd.for C15H21O4 + , 265.1434) that was appropriate for a molecular formula of C15H20O4.The NMR data of 5 showed that it shared the same trienedioic acid skeleton as epolactaene 4a [25].The main difference was the absence of a methyl in 5.The large vicinal coupling constant (J = 15.6 Hz) between H-6 (δH 6.23) and H-7 (δH 5.72) suggested an E-configuration for the ∆ 6 (7) double bond.In addition, the NOESY correlations between H3-1 (δH 1.65) and H-4 (δH 5.94), H-4 and H-6 and H2-9 (δH 2.45) and H3-15 (δH 1.74) indicated an E-configuration for the other double bonds (Figure 4).Therefore, the structure of 5, named as steckfusarin F, was elucidated .
The radical scavenging activities of all the isolated compounds were tested against DPPH; compounds 1, 2 and 4 showed radical scavenging activity against DPPH, with 37.3%, 54.3% and 45.8% inhibition at concentrations of 100 µg/mL, respectively.Due to the low mass of the active compounds, only compound 1 was measured for IC50.It had an   O 4 .The NMR data of 5 showed that it shared the same trienedioic acid skeleton as epolactaene 4a [25].The main difference was the absence of a methyl in 5.The large vicinal coupling constant (J = 15.6 Hz) between H-6 (δ H 6.23) and H-7 (δ H 5.72) suggested an E-configuration for the ∆ 6 (7) double bond.In addition, the NOESY correlations between H 3 -1 (δ H 1.65) and H-4 (δ H 5.94), H-4 and H-6 and H 2 -9 (δ H 2.45) and H 3 -15 (δ H 1.74) indicated an E-configuration for the other double bonds (Figure 4).Therefore, the structure of 5, named as steckfusarin F, was elucidated.
The radical scavenging activities of all the isolated compounds were tested against DPPH; compounds 1, 2 and 4 showed radical scavenging activity against DPPH, with 37.3%, 54.3% and 45.8% inhibition at concentrations of 100 µg/mL, respectively.Due to the low mass of the active compounds, only compound 1 was measured for IC 50 .It had an IC 50 value of 74.5 µg/mL, whereas ascorbic acid, used as a positive control, had an IC 50 value of 7.5 µg/mL.Furthermore, molecular docking analyses between the active compounds and superoxide dismutase (PDB ID: 7wx0) were performed to gain functional and structural insights (Figure 5).The results showed that compound 1 could interact with superoxide dismutase at the entrance of the catalytic pocket, with a calculated binding affinity of −6.3 kcal/mol.Compound 1 interacted with the residues SER-32 and GLN-153 via two hydrogen bonds, and five hydrophobic interactions were formed between compound 1 and the residues LYS-4, VAL-6, HIS-20, ALA-152 and GLN-153.The active site of 1 contained methoxy and carboxyl groups.The molecular docking results demonstrated that the negative binding free energy value between compound 2 and superoxide dismutase was −6.6 kcal/mol.Interaction modes revealed six hydrogen bonding interactions with the residues GLN-23, ARG-79, ARG-79, SER-102, LEU-103 and ILE-104.In addition, there were two hydrophobic interactions with LEU-103 and ALA-105 in the active site of superoxide dismutase.The active site of 2 contained hydroxyl and carboxyl groups and an epoxy ring.The size of compound 1 was 51 × 51 × 47, centered at x: 17.894, y: 7.9, z: 82.091.The size of compound 2 was 49 × 44 × 49, centered at x: 17.894, y: 7.9, z: 82.091.Compounds 1 and 2 had no π-π stacking and π-cation interactions.The docking studies suggested that compounds 1 and 2 could inhibit superoxide dismutase by tightly binding to catalytic amino acid residues through different types of interactions.
there were two hydrophobic interactions with LEU-103 and ALA-105 in the active site of superoxide dismutase.The active site of 2 contained hydroxyl and carboxyl groups and an epoxy ring.The size of compound 1 was 51 × 51 × 47, centered at x: 17.894, y: 7.9, z: 82.091.The size of compound 2 was 49 × 44 × 49, centered at x: 17.894, y: 7.9, z: 82.091.Compounds 1 and 2 had no π-π stacking and π-cation interactions.The docking studies suggested that compounds 1 and 2 could inhibit superoxide dismutase by tightly binding to catalytic amino acid residues through different types of interactions.Although many activities of fusarin derivatives have been reported [4,8,13,17], no obvious activities were detected for compounds 1-5.It was reported that the tetrahydropyran ring and methylation of the acid group from the sidechain were crucial to the activity of these compounds [21].However, none of the new compounds were methylated at C-19, which may be the reason why they did not display obvious activities.

General Experimental Procedures
Optical rotations were recorded using a Perkine Elmer 341 polarimeter (Hertford, UK).ECD spectra were recorded with a Chirascan circular dichroism spectrometer (Applied Photophysics, Surrey, UK).UV spectra were recorded on a UV-2600 UV-vis spectrophotometer (Shimadzu, Kyoto, Japan).The 1D and 2D NMR spectra were recorded on a Bruker AC 500 and 700 NMR (Broker, Fallanden, Switzerland) spectrometer with TMS as the internal standard.HRESIMS spectra were measured with a Bruker micro TOF-QII (Bruker, Fallanden, Switzerland) mass spectrometer in positive/negative ion mode.Silica gel GF-254 (10-40 mm) was used for thin-layer chromatography (TLC) (Qingdao Marine Chemical Factory, Qingdao, China).Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) and silica gel (200-300 mesh, 100-200 mesh) (Qingdao Marine Chemical Factory, Qingdao, China) were applied in column chromatography (CC).HPLC was carried out on a Hitachi Primaide with a YMC ODS Series column (YMC-Pack ODS-A, YMC Co. Ltd. (Kyoto, Japan), 250 × 10 mm i.d., S-5 µm, 12 nm).All solvents were analytical grade (Tianjin Fuyu Chemical and Industry Factory).The fermentation culture medium and reagents Although many activities of fusarin derivatives have been reported [4,8,13,17], no obvious activities were detected for compounds 1-5.It was reported that the tetrahydropyran ring and methylation of the acid group from the sidechain were crucial to the activity of these compounds [21].However, none of the new compounds were methylated at C-19, which may be the reason why they did not display obvious activities.

Fungal Strain
The fungal strain Penicillium steckii SCSIO 41040 was isolated from a green algae Botryocladia sp. that was collected from the South China Sea.The isolated fungal strain was stored on Muller Hinton broth (MB) agar (malt extract 16 g, agar 18 g, sea-salt 30 g, water 1 L and pH 7.4-7.8)slants at 4 • C and then deposited in CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, PR China.The ITS1-5.8S-ITS2 sequence region (550 base pairs (bp), GenBank accession no.OP349656) of strain SCSIO 41040 was amplified via the PCR process.DNA sequencing showed that it shared significant homology (100%) with Penicillium steckii (GenBank accession no.NR111488).

Fermentation and Extraction
A few loops of cells of the strain SCSIO 41040 were inoculated into a 500 mL Erlenmeyer flask containing 150 mL of seed medium (malt extract 1%, yeast extract 0.4%, glucose 0.4% and pH 7.2) and cultivated on a rotary shaker at 180 rpm and 28 • C for 48 h as a seed culture.Then, a large-scale fermentation of the fungal strain SCSIO 41040 was incubated at 25 • C in 1 L conical flasks containing a solid medium (300 mL/flask) composed of 200 g rice and 220 mL 3.2% (NaCl 3.2 g/H 2 O 100 mL) artificial seawater.After 32 days, the fermented material from each flask was extracted successively with EtOAc (700 mL/flask) and the combined EtOAc extract was suspended in MeOH and extracted with petroleum ether to remove rice oil.Finally, the MeOH solution was concentrated under reduced pressure to obtain a reddish-brown extract (56.0 g).

Isolation and Purification
The extract was subjected to silica gel column chromatography (CC), eluting with a gradient CH 2 Cl 2 -MeOH (100:0-0:100, v/v) to give 15 fractions based on TLC properties.Fr.

ECD Calculation
The relative configurations of the new compounds were subjected to random conformational searches using Spartan'14 and Gaussian 09 software with the Merck molecular force field (MMFF) and density functional theory (DFT)/TDDFT, respectively.The MMFF conformational search produced low-energy conformers with a Boltzmann population of more than 5%, which were geometrically optimized using the DFT method at the B3LYP/6-311G* level in MeOH using the IEFPCM model.For the stable conformers of the new compounds, the overall theoretical calculation of ECD was achieved in MeOH using timedependent density functional theory at the B3LYP/6-311G* level.We used the 0.2-0.4eV half bandwidth Multiwfn to generate the ECD spectra of different conformations and calculated the contribution according to the Boltzmann of each conformation after the UV correction [28].

Antimicrobial Assay
All new compounds were tested for antibacterial activity against Klebsiella pneumonia, Candida albicans Berkhout, Staphyloccocus aureus, Colletotrichum gloeosporioiles, Magnaporthe grisea and Clerotinia miyabeana Hanzawa.The tests were performed in 96-well plates using a modification of the broth microdilution method [30].

Anti-Inflammatory Assay
All new compounds were evaluated for their inhibitory activity against LPS-induced NF-κB activation in RAW264.7 cells using a luciferase reporter gene assay [31].The RAW264.7 cells, stably transfected with the NF-κB luciferase reporter gene, were plated in triplicate into 96-well plates for all treatments and controls.The compounds (20 µM) and BAY11-7082 (an NF-κB inhibitor used as a positive control (5 µM); Sigma-Aldrich, St. Louis, MO, USA) were used to pretreat the cells for 30 min, followed by stimulation with 5 µg/mL LPS for 8 h.The cells were harvested and luciferase activity measured using a luciferase assay system (Promega, Madison, WI, USA).

Cholesterol Transport Mechanism
Pancreatic triglyceride lipase (PTL) and Niemann-Pick C1-like 1 (NPC1L1) were the crucial targets involved in cholesterol cellar uptake.The inhibitory activity of the compounds against PTL was evaluated by colorimetry.Surface plasmon resonance (SPR) was used to analyze the binding of the compounds with NPC1L1, and the activities of targeted compounds for NPC1L1 were studied [32,33].

PFKFB3 Kinase Inhibitory Activity
PFKFB3 kinase inhibitory activity was measured using an ADP-Glo Kinase Assay kit (Promega) according to a published modified protocol [34].Compound solution (1 µL, with a final concentration of 20 µM in 1% DMSO) and 2 µL enzyme solution were added to 384-well plates, followed by incubation at room temperature (RT) for 30 min.After 2 h incubation at RT, 4 µL ADP-Glo Reagent was added and then the incubation continued for 1 h prior to the addition of 8 µL Kinase Detection Reagent and a further incubation of 1 h.The luminous signal was measured using an Envision flat panel reader (PerkinElmer, Waltham, MA, USA).

PI3K Kinase Inhibitory Activity
An amount of 0.5 µL of the compounds was preincubated with 14.5 µL of enzyme and PIP2 substrate for 10 min before addition of 5 µL of ATP to achieve a final ATP concentration of 10 µM [35].The total reaction volume was 20 µL, and the reaction was allowed to proceed for 45 min at room temperature before the addition of the stop solution and detection mixture provided in the kit.The luminous signal was measured using an Envision flat panel reader (PerkinElmer).

Measurement of AChE Inhibition Activity
We evaluated the AChE enzyme inhibitory activity of compounds according to the slightly modified spectrophotometric method [36].Tacrine was used as a positive control.

Figure 2 .
Figure 2. Experimental and calculated ECD spectra for compounds 1 and 2.

Figure 5 .
Figure 5. Low-energy binding conformations of 1 and 2 bound to superoxide dismutase (generated by molecular docking).

. 4a 4b δ C , Type δ H (J in Hz) δ C , Type δ H (J in Hz)
Compound 5 was obtained as a yellow oil.It showed a [M + H] + ion peak at m/z 265.1431 in the positive-ion HRESIMS (calcd.for C 15 H 21 O 4 + , 265.1434) that was appropriate for a molecular formula of C 15 H 20