Prenylated Phenol and Benzofuran Derivatives from Aspergillus terreus EN-539, an Endophytic Fungus Derived from Marine Red Alga Laurencia okamurai

Three new prenylated phenol derivatives, terreprenphenols A–C (1–3), along with four known related compounds (4–7), were isolated from Aspergillus terreus EN-539, an endophytic fungus obtained from the marine red alga Laurencia okamurai. The structures of these compounds were established by extensive analysis of 1D/2D NMR data, mass spectrometric data, and optical rotation (OR). The corresponding relationship between absolute configuration and optical rotation for known compounds anodendroic acid (4) and asperterreusine C (5) was ambiguous in literature, and their absolute configurations were therefore discussed and confirmed for the first time by time-dependent density functional (TDDFT) ECD and OR calculations. Compounds 1–7 inhibited some common aquatic bacteria with MIC values ranging from 2 to 64 μg/mL.


Introduction
The growing trend in the discovery of new marine natural products from marine microorganisms has continued increasing over the last few years and represented 57% of the total new marine natural products reported in 2017 [1]. However, the rare occurrence of novel compounds and chemical rediscovery have been chronic problems in microbial secondary metabolites research [2,3]. Aspergillus terreus is a species complex currently comprised of 14 cryptic species, which is found worldwide in the environment [4]. In our previous research on new bioactive secondary metabolites from marine-derived fungi [5][6][7][8], a fungal strain Aspergillus terreus EN-539 was isolated from marine red alga Laurencia okamurai, which has been reported as a prolific producer of halogenated organic molecules, such as sesquiterpenes and nonterpenoid C 15 -acetogenins [9]. Chemical investigations were performed on the culture extracts of the marine fungus A. terreus EN-539. Two new meroterpenoids, aperterpenes N and O, along with related derivatives have been reported from A. terreus EN-539, which was cultured on rice-solid medium [7]. To enhance the chemical diversity of secondary metabolites, the fungal strain A. terreus EN-539 was further cultivated on MH2 medium, which resulted in the production of different metabolites compared with that culturing on rice-solid medium, as evidenced by HPLC analysis. As a result, three new prenylated phenol derivatives including terreprenphenol A (1), terreprenphenol B (2), and terreprenphenol C (3), along with four known related compounds (4)(5)(6)(7), were isolated and identified. known related compounds (4)(5)(6)(7), were isolated and identified. Moreover, as the corresponding relationship between stereochemistry and optical rotation (OR) for known compounds, anodendroic acid (4) and asperterreusine C (5), were ambiguous in literature, their absolute configurations were discussed and confirmed on the basis of time-dependent density functional (TDDFT)-ECD and OR calculations. The antimicrobial activities against some common aquatic bacteria, as well as antioxidative activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, were evaluated. This paper describes the isolation, characterization, and bioactivities of compounds 1-7 ( Figure 1).

Structure Elucidation of the New Compounds
Terreprenphenol A (1) was obtained as a colourless solid, and its formula was determined as C13H16O3 on the basis of HRESIMS data ( Figure S1), indicating six degrees of unsaturation. The 1 H and 13 C NMR spectroscopic data of 1 indicated the presence of a 1,3,4-trisubstituted benzene ring, two methyls, two methylenes (including one oxygenated), one olefinic methine, and two nonprotonated (including one ketone and one olefinic) carbon atoms. Detailed analysis of the 1 H and 13 C NMR data (Table 1) revealed that 1 is a prenylated phenol derivative similar to 4-hydroxy-3-(3methyl-2-butenyl)acetophenone (HMBA, 8), which is the main secondary metabolite of Senecio nutans (Asteraceae), a medicinal plant of northwestern Argentina [10,11]. However, the resonances for one methyl at δH 2.58 and δC 26.1 in the 1 H and 13 C NMR spectra of HMBA were not present in those of 1. Instead, signals for an oxygenated methylene were observed at δH 4.63 (H-8) and δC 64.3 (C-8) in the 1 H and 13 C NMR spectra of 1. These spectroscopic features suggested that compound 1 was the 8hydroxylated derivative of HMBA. The COSY and HMBC data ( Figure 2) supported the above deduction. The structure of 1 was thus determined and it was named as terreprenphenol A. Terreprenphenol B (2) was obtained as a white, amorphous powder with the molecular formula C12H14O4 as established by HRESIMS data (Figure S7), indicating six degrees of unsaturation. Signals for a 1,3,4-trisubstituted benzene ring were also observed at δH 6.69 (d, J = 8.3 Hz, H-5), 7.70 (d, J = 8.3 Hz, H-6), and 7.71 (s, H-2) in the 1 H NMR spectrum of 2, indicating that it could be another prenylated phenol derivative. The 1 H and 13 C NMR data (Table 1) of 2 were very similar to those of 4-hydroxy-3-prenybenzoic acid (6), a known benzoic acid derivative isolated from the dogwood anthracnose fungus Discula sp. by Venkatasubbaiah et al. [12]. However, the resonances of two olefinic carbons

Structure Elucidation of the New Compounds
Terreprenphenol A (1) was obtained as a colourless solid, and its formula was determined as C 13 H 16 O 3 on the basis of HRESIMS data ( Figure S1), indicating six degrees of unsaturation. The 1 H and 13 C NMR spectroscopic data of 1 indicated the presence of a 1,3,4-trisubstituted benzene ring, two methyls, two methylenes (including one oxygenated), one olefinic methine, and two nonprotonated (including one ketone and one olefinic) carbon atoms. Detailed analysis of the 1 H and 13 C NMR data (Table 1) revealed that 1 is a prenylated phenol derivative similar to 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone (HMBA, 8), which is the main secondary metabolite of Senecio nutans (Asteraceae), a medicinal plant of northwestern Argentina [10,11]. However, the resonances for one methyl at δ H 2.58 and δ C 26.1 in the 1 H and 13 C NMR spectra of HMBA were not present in those of 1. Instead, signals for an oxygenated methylene were observed at δ H 4.63 (H-8) and δ C 64.3 (C-8) in the 1 H and 13 C NMR spectra of 1. These spectroscopic features suggested that compound 1 was the 8-hydroxylated derivative of HMBA. The COSY and HMBC data ( Figure 2) supported the above deduction. The structure of 1 was thus determined and it was named as terreprenphenol A.
Mar. Drugs 2019, 17, x 2 of 9 known related compounds (4-7), were isolated and identified. Moreover, as the corresponding relationship between stereochemistry and optical rotation (OR) for known compounds, anodendroic acid (4) and asperterreusine C (5), were ambiguous in literature, their absolute configurations were discussed and confirmed on the basis of time-dependent density functional (TDDFT)-ECD and OR calculations. The antimicrobial activities against some common aquatic bacteria, as well as antioxidative activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, were evaluated. This paper describes the isolation, characterization, and bioactivities of compounds 1-7 ( Figure 1).

Structure Elucidation of the New Compounds
Terreprenphenol A (1) was obtained as a colourless solid, and its formula was determined as C13H16O3 on the basis of HRESIMS data ( Figure S1), indicating six degrees of unsaturation. The 1 H and 13 C NMR spectroscopic data of 1 indicated the presence of a 1,3,4-trisubstituted benzene ring, two methyls, two methylenes (including one oxygenated), one olefinic methine, and two nonprotonated (including one ketone and one olefinic) carbon atoms. Detailed analysis of the 1 H and 13 C NMR data (Table 1) revealed that 1 is a prenylated phenol derivative similar to 4-hydroxy-3-(3methyl-2-butenyl)acetophenone (HMBA, 8), which is the main secondary metabolite of Senecio nutans (Asteraceae), a medicinal plant of northwestern Argentina [10,11]. However, the resonances for one methyl at δH 2.58 and δC 26.1 in the 1 H and 13 C NMR spectra of HMBA were not present in those of 1. Instead, signals for an oxygenated methylene were observed at δH 4.63 (H-8) and δC 64.3 (C-8) in the 1 H and 13 C NMR spectra of 1. These spectroscopic features suggested that compound 1 was the 8hydroxylated derivative of HMBA. The COSY and HMBC data ( Figure 2) supported the above deduction. The structure of 1 was thus determined and it was named as terreprenphenol A. Terreprenphenol B (2) was obtained as a white, amorphous powder with the molecular formula C12H14O4 as established by HRESIMS data ( Figure S7), indicating six degrees of unsaturation. Signals for a 1,3,4-trisubstituted benzene ring were also observed at δH 6.69 (d, J = 8.3 Hz, H-5), 7.70 (d, J = 8. 3 Hz, H-6), and 7.71 (s, H-2) in the 1 H NMR spectrum of 2, indicating that it could be another prenylated phenol derivative. The 1 H and 13 C NMR data (Table 1) of 2 were very similar to those of 4-hydroxy-3-prenybenzoic acid (6), a known benzoic acid derivative isolated from the dogwood anthracnose fungus Discula sp. by Venkatasubbaiah et al. [12]. However, the resonances of two olefinic carbons  (Table 1) of 2 were very similar to those of 4-hydroxy-3-prenybenzoic acid (6), a known benzoic acid derivative isolated from the dogwood anthracnose fungus Discula sp. by Venkatasubbaiah et al. [12]. However, the resonances of two olefinic carbons (δ C 122.8, C-2'; 132.2, C-3') in the 13 C NMR spectrum of 4-hydroxy-3-prenybenzoic acid (6) were replaced by resonances of two oxygenated carbons (δ C 68.6, C-2'; 76.6, C-3') in that of 2. The HRESIMS as well as COSY and HMBC data of 2 suggested that the double bond at C-2' and C-3' in 6 was converted into an epoxide ring in 2.   Terreprenphenol C (3) was obtained as a colourless solid, and its formula was determined as C 12 H 14 O 3 , with one oxygen atom less than 2, on the basis of HRESIMS data ( Figure S13). Comprehensive analysis of the 1 H and 13 C NMR data (Table 1) of 3 showed a structurally close relationship to that of 2, except that the carbonyl group at C-7 (δ C 173.4) in 2 was replaced by an aldehyde group (δ C 191.9; δ H 10.64, s) in 3. The planar structure of 3 was further confirmed by the COSY and HMBC data ( Figure 2). The absolute configuration of 3 was also determined as 2'S on the basis of its optical rotation value of and 2. Moreover, it should be mentioned that the benzoate of 3, methyl 3-((3,3-dimethyloxiran-2yl)methyl)-4-hydroxybenzoate, was reported as an intermediate in the syntheses of benzofuran derivatives hostmaniene, 5-formyl-2-(isopropyl-1'-ol)benzofuran, and anadenfroic acid, without purification and identification [14].
Anodendroic acid (4) and asperterreusine C (5) were isolated and identified by comparing their NMR spectroscopic data (Figures S19-S22) and optical rotations with those reported in the literatures [14][15][16][17]. However, the relationship between absolute configuration and optical rotation for the known compounds anodendroic acid (4) and asperterreusine C (5) was ambiguous in the literature, and their absolute configurations were therefore discussed and confirmed by TDDFT-ECD and OR calculations. Anodendroic acid (4) was first isolated from the higher plant Anodendron affine Durce with optical rotation of [α] 26 D = −19 (c 0.7, EtOH) [15], and then its absolute configuration was determined as being R by chemical synthesis, with optical rotation of [α] 15 D = −35.2 (c 0.682, EtOH) [18]. Recently, the (+)-S-anodendroic acid was isolated and identified from another higher plant Euodia lepta, with an optical rotation of [α] 25 D = +42.0 (c 0.15, EtOH) [16]. Quadricinctafuran A, possessing the same planar structure as that of anodendroic acid, was established as having an R configuration on the basis of X-ray crystallographic analysis by Prompanya et al. [19]. However, the optical rotation of quadricinctafuran A was measured as [α] 20 D = +74 (c 0.03, MeOH) (R configuration with positve OR and S configuration with negative OR), inconsistent with the corresponding relationship between sterechemistry and optical rotation (R configuration with negative OR, S configuration with positive OR) in other literature [14][15][16]. Considering the ambiguity, the absolute configuration of 4 was thus established by the TDDFT-ECD calculation in Gaussian 09 [20]. We obtained the minimum energy conformers by geometry optimization of S and R isomers of 4, and then employed the TDDFT method at the B3LYP/6-31G level to obtain a calculated ECD spectra of 4. The experimental ECD spectrum of 4 exhibited excellent accordance with that calculated for R isomer of 4 and was opposite to that calculated for an S isomer of 4, which allowed unambiguous assignment of its absolute configuration (Figure 3).  [16]. Quadricinctafuran A, possessing the same planar structure as that of anodendroic acid, was established as having an R configuration on the basis of X-ray crystallographic analysis by Prompanya et al. [19]. However, the optical rotation of quadricinctafuran A was measured as [α] 20 D = +74 (c 0.03, MeOH) (R configuration with positve OR and S configuration with negative OR), inconsistent with the corresponding relationship between sterechemistry and optical rotation (R configuration with negative OR, S configuration with positive OR) in other literature [14][15][16]. Considering the ambiguity, the absolute configuration of 4 was thus established by the TDDFT-ECD calculation in Gaussian 09 [20]. We obtained the minimum energy conformers by geometry optimization of S and R isomers of 4, and then employed the TDDFT method at the B3LYP/6-31G level to obtain a calculated ECD spectra of 4. The experimental ECD spectrum of 4 exhibited excellent accordance with that calculated for R isomer of 4 and was opposite to that calculated for an S isomer of 4, which allowed unambiguous assignment of its absolute configuration ( Figure 3).  0.51, MeOH), respectively, which satisfied the reported correspondances between sterechemistry and optical rotations (R configuration with negative OR and S configuration with positive OR) for anodendroic acid [18], (+)-S-anodendroic acid [16], asperterreusine C [17], and (S)-5formyl-2-(isopropyl-1′-ol)-2,3-dihydrobenzofuran [21]. This corresponding relationship was further verified by OR calculation at the B3LYP/6-31G(d) level in Gaussian 09 [20]. The OR calculation for compounds 4 and 5 (Table 2) indicated that the R configuration was consistent with negative OR and the S configuration was featured with the opposite sign, which established the absulute configurations of 4 and 5 by comparing their measured OR values. This matched well with the results of the TDDFT-ECD calculation.
In addition to compounds 1-5, the known prenylated phenol derivatives, 4-hydroxy-3prenybenzoic acid (6) and 4-hydroxy-3-(3-methyl-but-2-enyl)-benzaldehyde (7), were isolated and identified from the MH2 culture extract of fungal strain A. terreus EN-539. Their structures were , respectively, which satisfied the reported correspondances between sterechemistry and optical rotations (R configuration with negative OR and S configuration with positive OR) for anodendroic acid [18], (+)-S-anodendroic acid [16], asperterreusine C [17], and (S)-5-formyl-2-(isopropyl-1 -ol)-2,3-dihydrobenzofuran [21]. This corresponding relationship was further verified by OR calculation at the B3LYP/6-31G(d) level in Gaussian 09 [20]. The OR calculation for compounds 4 and 5 (Table 2) indicated that the R configuration was consistent with negative OR and the S configuration was featured with the opposite sign, which established the absulute configurations of 4 and 5 by comparing their measured OR values. This matched well with the results of the TDDFT-ECD calculation.

Biological Activities of the Isolated Compounds
Compounds 1-7 were assayed for their antioxidative activity against DPPH radical. The result showed that these prenylated phenol and benzofuran derivatives displayed no observed DPPH radical scavenging activity, except that compound 7 exhibited weak scavenging activity with IC 50 value of 0.9 mM. Butylated hydroxytoluene (BHT) was measured as the positive control for DPPH radical scavenging activity (IC 50 , 72 µM).

Fungal Material
The isolation and identification of the fungal material A. terreus EN-539 were the same as those reported in our previous publications [7]. The strain is preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS).

Fermentation
For chemical investigations, the fresh mycelia of fungal strain A. terreus EN-539 was cultured on PDA (Potato Dextrose Agar) medium at 28°C for seven days, and then inoculated into 1 L conical flasks containing 300 mL of MH2 broth medium (sucrose 2%, mannitol 2%, yeast extract 0.3%, peptone 0.5%, K 2 HPO 4 0.05%, and MgSO 4 ·7H 2 O 0.03% in seawater, which was naturally sourced and filtered from the Huiquan Gulf of the Yellow Sea near the campus of IOCAS, pH 6.5-7.0) for 30 days at room temperature.

Extraction and Isolation
The whole fermented cultures (100 flasks, 30 L) were filtered to separate the broth from the mycelia. The former was extracted four times with ethyl acetate (EtOAc), while the mycelia was extracted four times with a mixture of 80% acetone and 20% H 2 O. The acetone solution was evaporated under reduced pressure to develop an aqueous solution, which was then extracted four times with EtOAc. The two EtOAc solutions were combined and concentrated under reduced pressure to give an extract (48 g), on the basis of TLC and HPLC analysis, which was fractionated by silica gel vacuum liquid chromatography (VLC) using different solvents of increasing polarity from petroleum ether (PE) to MeOH to yield 9 fractions (Frs. 1-9). Fr. 4 (4.5 g), eluted with PE-EtOAc (2:1), was further purified by reversed-phase column chromatography (CC) over Lobar LiChroprep RP-18 with a MeOH-H 2 O gradient (from 10:90 to 100, v/v) to yield 10 subfractions.

Computational Section
Conformational searches were performed via molecular mechanics using the MM+ method in HyperChem software (Version 8.0, Hypercube, Inc., Gainesville, FL, USA), and the geometries were further optimized at the B3LYP/6-31G(d) level via Gaussian 09 software (Version D.01; Gaussian, Inc.:Wallingford, CT, USA) [20] to give the energy-minimized conformers. Then, the optimized conformers were subjected to the calculations of ECD and OR by using TDDFT at B3LYP/6-31G level. Solvent effects of the MeOH solution were evaluated at the same DFT level using the SCRF/PCM method.

Conclusions
In summary, chemical investigations were performed on the marine fungus A. terreus EN-539. Two new meroterpenoids, aperterpenes N and O, along with related derivatives have been reported from A. terreus EN-539, which was cultured on rice-solid medium [7]. The coculture of A. terreus EN-539 and the symbiotic fungus Paecilomyces lilacinus EN-531 induced the production of a new terrein derivative, namely asperterrein and a known dihydroterrein, which were not detected in the axenic cultures of both strains [25]. To enhance the chemical diversity of secondary metabolites, A. terreus EN-539 was further cultivated on MH2 medium, which resulted in the production of three new prenylated phenol derivatives including terreprenphenol A (1), terreprenphenol B (2), and terreprenphenol C (3), along with four known related compounds (4-7). The absolute configurations of benzofuran derivatives (4 and 5) were discussed and confirmed on the basis of TDDFT-ECD and OR calculations. Compounds 1, 6, and 7 showed broad-spectrum inhibitory activity against the pathogenic bacteria in the assay with MIC values ranging from 2 to 64 µg/mL, which might be used as potential molecules in the development of drug leads, or modified to find more active derivatives for the treatment of microbial infection in aquaculture.