Uncommon Polyketides from Penicillium steckii AS-324, a Marine Endozoic Fungus Isolated from Deep-Sea Coral in the Magellan Seamount

Four unusual steckwaic acids E–H (1–4), possessing a rarely described acrylic acid unit at C-4 (1–3) or a double bond between C-12 and C-13 (4) are reported for the first time, along with four new analogues (5–8) and two known congeners (9 and 10). They were purified from the organic extract of Penicillium steckii AS-324, an endozoic fungus obtained from a deep-sea coral Acanthogorgiidae sp., which was collected from the Magellan Seamount at a depth of 1458 m. Their structures were determined by the interpretation of NMR and mass spectroscopic data. The relative and absolute configurations were determined by NOESY correlations, X-ray crystallographic analysis, and ECD calculations. All compounds were tested for their antimicrobial activities against human- and aquatic-pathogenic bacteria and plant-related pathogenic fungi.


Introduction
Deep-sea-living organisms have evolved under extreme environmental conditions, which have influenced the development of various biochemical functions compared to those living in shallow-water organisms [1,2]. Although the area of deep-sea habitats is much larger than that of shallow-sea habitats, compounds isolated from deep-sea organisms accounted for only~2% of the more than 30,000 marine natural products [3,4], whereas approximately 75% of these molecules exhibited notable bioactivities. Improved technological capacity for sampling from the deep-sea environment has improved the discovery of deep-sea-derived natural products. In recent years, the number of deep-seasourced natural products increased rapidly, and these compounds usually display high bioactivity hits in bioassays [5]. Although the nature of the associations between a host and its associated microbes is far from understood, there is growing evidence that some coral-associated fungi have adopted the ability to produce secondary metabolites that are structurally divergent from their terrestrial counterparts [6][7][8]. Marine animal-related fungi often produce bioactive metabolites that might be interpreted as chemically mediated defense mechanisms to protect their host organisms from environmental hazards such as predation and pathogenic invasion [9]. Some studies have reported on the isolation of marine coral-associated Penicillium spp. as producers of bioactive metabolites [10][11][12].

Results
The culture of P. steckii AS-324 was extracted with EtOAc to gain the organic extract, which was then fractionated and purified by various chromatographic methods to yield compounds 1-10 ( Figure 1).
Steckwaic acid E (1) was initially obtained as a white amorphous powder, and the molecular formula was assigned as C16H24O3 by negative HRESIMS data. The 1 H and 13 C NMR data (Table 1) revealed the presence of three doublet methyl substitutions, one methylene, eleven methines (with one oxygenated and four olefinic), and one carboxyl carbon. A large spin system incorporating H-2 through H-13 and three methyls, H3-14, H3-15, and H3-16, confirmed the presence of a decalin skeleton with three methyl-substitution at C-6, C-8, and C-13 ( Figure 2). HMBC correlations from H-3 to C-1, C-5, and C-13 and from H-2 to C-4 indicated the position of one acrylic acid side chain at C-4 ( Figure 2). The large coupling constant between H-2 and H-3 (J = 15. 4 Hz) suggested the E-geometry of the double bond, whereas the smaller coupling constant between H-11 and H-12 (J = 9. 8 Hz) revealed the Z-geometry. Thus, the planar structure of 1 was determined. The coupling constants between H-4 and H-5, between H-5 and H-10, and between H-8 and H-9, as well as between H-9 and H-10, were all 9.5 Hz, indicating the trans-orientation of these adjacent proton pairs in the cyclohexane/cyclohexene units. NOESY correlations from H-3 to H-5 and H3-15, as well as from H-5 to H-9, demonstrated them on the same face of these

Results
The culture of P. steckii AS-324 was extracted with EtOAc to gain the organic extract, which was then fractionated and purified by various chromatographic methods to yield compounds 1-10 ( Figure 1).
Steckwaic acid E (1) was initially obtained as a white amorphous powder, and the molecular formula was assigned as C 16 H 24 O 3 by negative HRESIMS data. The 1 H and 13 C NMR data (Table 1) revealed the presence of three doublet methyl substitutions, one methylene, eleven methines (with one oxygenated and four olefinic), and one carboxyl carbon. A large spin system incorporating H-2 through H-13 and three methyls, H 3 -14, H 3 -15, and H 3 -16, confirmed the presence of a decalin skeleton with three methyl-substitution at C-6, C-8, and C-13 ( Figure 2). HMBC correlations from H-3 to C-1, C-5, and C-13 and from H-2 to C-4 indicated the position of one acrylic acid side chain at C-4 ( Figure 2). The large coupling constant between H-2 and H- 3 (J = 15.4 Hz) suggested the E-geometry of the double bond, whereas the smaller coupling constant between  revealed the Z-geometry. Thus, the planar structure of 1 was determined. The coupling constants between H-4 and H-5, between H-5 and H-10, and between H-8 and H-9, as well as between H-9 and H-10, were all 9.5 Hz, indicating the trans-orientation of these adjacent proton pairs in the cyclohexane/cyclohexene units. NOESY correlations from H-3 to H-5 and H 3 -15, as well as from H-5 to H-9, demonstrated them on the same face of these protons, while correlations from H-10 to H-6, H-8, and H-13 placed them on the opposite side ( Figure 3).  6.84, dd (15.4, 10.9) 154.2, CH 6.88, dd (15.3, 10.9) 153.2, CH 6.86, dd (15.4, 10.9) 4 48.0, CH 2.46,ddd (10.9,9.5,5.6) 47.5,CH 2.46,td (10.9,5. protons, while correlations from H-10 to H-6, H-8, and H-13 placed them on the opposite side ( Figure 3). 154.2, CH 6.84, dd (15.4, 10.9) 154.2, CH 6.88, dd (15.3, 10.9) 153.2, CH 6.86, dd (15.4, 10.9) 4 48.0, CH 2.46, ddd (10.9, 9.5, 5.6)    To unambiguously confirm the structure and configuration of compound 1, attempts to cultivate quality crystals were performed, and suitable crystals were obtained by dissolving the samples in MeOH and refrigerating them to evaporate the solvent slowly. Single-crystal X-ray diffraction analysis using Cu Kα radiation confirmed the structure of 1, and the absolute configuration was 4S, 5R, 6R, 8S, 9R, 10R, and 13S with the Flack parameter −0.09 (13) (Figure 4). To unambiguously confirm the structure and configuration of compound 1, attempts to cultivate quality crystals were performed, and suitable crystals were obtained by dissolving the samples in MeOH and refrigerating them to evaporate the solvent slowly. Single-crystal X-ray diffraction analysis using Cu Kα radiation confirmed the structure of 1, and the absolute configuration was 4S, 5R, 6R, 8S, 9R, 10R, and 13S with the Flack parameter −0.09 (13) (Figure 4).
Steckwaic acid F (2), obtained as colorless crystals, was assigned a molecular formula of C16H22O4 according to the HRESIMS analysis. Detailed inspection of its NMR data revealed the same skeleton as that of compound 1, and the main differences are that the oxygenated methine resonating at δC/δH 77.0/2.59 (CH-9) in 1 was replaced by methylene resonating at δC/δH 35.4/1.93 and 1.11 (CH2-9) in 2, whereas a carboxyl carbon resonating at δC 176.9 (C-16) was observed by HMBC in 2, which replaced the methyl group resonating at δC/δH 19.2/0.91 (CH3-16) in 1. The large spin system similar to that in 1 was otherwise intact, albeit with some chemical shift differences and the absence of the methyl group CH3-16 in 2 ( Figure 2). HMBC correlations from H-8 and H-9 to the carboxyl carbon C-16, as well as from H-9 to C-5 and C-7, confirmed the planar structure of compound 2 ( Figure 2). The coupling constant between H-2 and H-3 (J = 15. 3 Hz) suggested the E-geometry of the double bond. NOE correlations from H-3 to H-5, H3-14, and H3-15 suggested the protons were in the same orientation, whereas correlations from H-6 to H-4 and H-8 as well as from H-8 to H-10 positioned them on the other side ( Figure 3). The structure and absolute configuration of compound 2 were confirmed by X-ray diffraction analysis using Cu Kα radiation, and the Flack parameter 0.05(10) permitted assignment of the absolute configuration as 4S, 5S, 6R, 8S, 10R, and 13S ( Figure 4).  Steckwaic acid G (3), obtained as a white amorphous powder, was assigned the molecular formula C16H24O3 by HRESIMS and NMR data. The chemical structure of compound 3 was almost identical to that of 2. The main difference between 3 and 2 was evident in the nonappearance of one of the two carboxyl carbons C-16 (δC 176.9 in 2) in the 13 C-NMR spectrum of 3, the appearance of 1 H-and 13 C-NMR resonances for an oxymethylene moiety CH2-16 (δC 66.3 and δH 3.20 in 3), and the related changes in chemical shifts and multiplicities of nearby carbons and protons around CH-9 (Table 1). This spectral evidence suggests that the carboxyl carbon C-16 in 2 was reduced to form an oxymeth- Steckwaic acid F (2), obtained as colorless crystals, was assigned a molecular formula of C 16 H 22 O 4 according to the HRESIMS analysis. Detailed inspection of its NMR data revealed the same skeleton as that of compound 1, and the main differences are that the oxygenated methine resonating at δ C /δ H 77.0/2.59 (CH-9) in 1 was replaced by methylene resonating at δ C /δ H 35.4/1.93 and 1.11 (CH 2 -9) in 2, whereas a carboxyl carbon resonating at δ C 176.9 (C-16) was observed by HMBC in 2, which replaced the methyl group resonating at δ C /δ H 19.2/0.91 (CH 3 -16) in 1. The large spin system similar to that in 1 was otherwise intact, albeit with some chemical shift differences and the absence of the methyl group CH 3 -16 in 2 ( Figure 2). HMBC correlations from H-8 and H-9 to the carboxyl carbon C-16, as well as from H-9 to C-5 and C-7, confirmed the planar structure of compound 2 ( Figure 2). The coupling constant between H-2 and H-3 (J = 15. 3 Hz) suggested the E-geometry of the double bond. NOE correlations from H-3 to H-5, H 3 -14, and H 3 -15 suggested the protons were in the same orientation, whereas correlations from H-6 to H-4 and H-8 as well as from H-8 to H-10 positioned them on the other side ( Figure 3). The structure and absolute configuration of compound 2 were confirmed by X-ray diffraction analysis using Cu Kα radiation, and the Flack parameter 0.05(10) permitted assignment of the absolute configuration as 4S, 5S, 6R, 8S, 10R, and 13S ( Figure 4).
Steckwaic acid G (3), obtained as a white amorphous powder, was assigned the molecular formula C 16 H 24 O 3 by HRESIMS and NMR data. The chemical structure of compound 3 was almost identical to that of 2. The main difference between 3 and 2 was evident in the nonappearance of one of the two carboxyl carbons C-16 (δ C 176.9 in 2) in the 13 C-NMR spectrum of 3, the appearance of 1 H-and 13 C-NMR resonances for an oxymethylene moiety CH 2 -16 (δ C 66.3 and δ H 3.20 in 3), and the related changes in chemical shifts and multiplicities of nearby carbons and protons around CH-9 (Table 1). This spectral evidence suggests that the carboxyl carbon C-16 in 2 was reduced to form an oxymethylene unit in 3, and this assignment was further confirmed by COSY and HMBC correlations ( Steckwaic acid H (4) was determined to have the molecular formula C18H26O3 based on the negative HRESIMS data. Its NMR data ( Table 2) displayed typical signals of a decalin skeleton with three methyl signals for C-8, C-10, and C-15, and the appropriately modified acrylic acid substituent in 1-3 was changed to a penta-2,4-dienoic acid moiety in 4. HMBC correlations from the olefinic proton H-13 to C-7, C-11, and C-15, as well as from H-6 and H-14 to the nonprotonated olefinic carbon C-12, were located in the position of a double bond between C-12 and C-13. In addition, HMBC correlations from the oxymethine proton H-14 to C-6, C-12, and C-18 confirmed the hydroxy-substituent at C-14.  Steckwaic acid H (4) was determined to have the molecular formula C 18 H 26 O 3 based on the negative HRESIMS data. Its NMR data ( Table 2) displayed typical signals of a decalin skeleton with three methyl signals for C-8, C-10, and C-15, and the appropriately modified acrylic acid substituent in 1-3 was changed to a penta-2,4-dienoic acid moiety in 4. HMBC correlations from the olefinic proton H-13 to C-7, C-11, and C-15, as well as from H-6 and H-14 to the nonprotonated olefinic carbon C-12, were located in the position of a double bond between C-12 and C-13. In addition, HMBC correlations from the oxymethine proton H-14 to C-6, C-12, and C-18 confirmed the hydroxy-substituent at C-14. Large coupling constants between H-2 and H-3 (J = 15.3 Hz), as well as H-4 and H-5 (J = 15.1 Hz), showed the E-geometry of two double bonds at C-2 and C-4. The relative configuration of 4 was determined by a NOESY spectrum. NOE correlations from H-5 to H-7, H-14, H 3 -16, and H 3 -17 placed them on the same face of the molecule, whereas correlations from H-8 to H-10 indicated these protons were on the other side. To clarify the absolute configuration of 4, the ECD spectra of minimum energy conformers by the TDDFT method at BH&HLYP/TZVP and CAM-B3LYP/TZVP levels were calculated, and the experimental ECD spectrum of 4 matched well with that of the calculated spectrum for (6R, 7R, 8R, 10S, 14S, and 15R)-4 ( Figures 6 and S59).  Compound 5 was obtained as a white amorphous powder, and the molecular formula was assigned as C19H28O4 by HRESIMS data. Detailed analysis of the 1 H and 13 C data ( Table 2) showed that it was similar to tanzawaic acid U [17] except that resonances for the methine unit at δC 32.3 and δH 1.46 (CH-10) in tanzawaic acid U were replaced by an oxygenated/nonprotonated carbon at δC 68.0 (C-10) in 5. These observations were further supported by relevant COSY and HMBC correlations (Figure 2). The coupling constants of two double bonds at C-2 and C-4 were 15.2 and 15.3 Hz, respectively, suggesting Egeometry for the double bonds. NOE correlations from H-5 to H-7 and H3-17, as well as Compound 5 was obtained as a white amorphous powder, and the molecular formula was assigned as C 19 H 28 O 4 by HRESIMS data. Detailed analysis of the 1 H and 13 C data ( Table 2) showed that it was similar to tanzawaic acid U [17] except that resonances for the methine unit at δ C 32.3 and δ H 1.46 (CH-10) in tanzawaic acid U were replaced by an oxygenated/nonprotonated carbon at δ C 68.0 (C-10) in 5. These observations were further supported by relevant COSY and HMBC correlations (Figure 2). The coupling constants of two double bonds at C-2 and C-4 were 15.2 and 15.3 Hz, respectively, suggesting Egeometry for the double bonds. NOE correlations from H-5 to H-7 and H 3 -17, as well as from H 3 -17 to H 3 -18, indicated the same orientation of these protons, while correlations from H-6 to H-12 and H-13 showed them on the opposite side. The absolute configuration of 5 was determined by both ECD calculation and comparisons with known compounds 9 and 10. The experimental ECD spectrum of 5 aptly matched the calculated spectra of (6R, 7R, 8R, 10R, 12S, 13S)-5 at BH&HLYP/TZVP, CAM-B3LYP/TZVP, and PBE0/TZVP levels ( Figure 7 and Figure S59). Besides, 5 showed a positive Cotton effect at approximately 264 nm similar to that of compounds 9 and 10 ( Figure 8), which also confirmed the absolute configuration of 5. Compound 5 was named 10-hydroxytanzawaic acid U.  Compound 6 was isolated as a white amorphous powder and assigned the molecular formula C20H28O5 by negative HRESIMS data. A detailed comparison of the NMR spectral data (Table 2) with the known compound tanzawaic acid R [17] suggested that they were very similar. However, signals for a carboxyl carbon at δC 170.4 and a methyl group at δC/δH 20.7/2.00 related to an acetoxyl group were observed in the NMR spectra of 6. HMBC correlations from H2-18 to C-9, C-11, and C-19 as well as H3-20 to C-19 placed the acetoxyl group at C-18 ( Figure 2). Large coupling constants between H-2 and H-3, as well as between H-4 and H-5 (J = 15.3 Hz), indicated the E-geometry of two double bonds. NOESY correlations from H3-17 to H-5, H-7, and H2-18, as well as from H2-18 to H-11β, suggested the same orientation of these protons, while correlations from H-12 to H-8 and H-10 as well as from H-13 to H-11α placed these groups on the opposite face. From the ECD data and biogenetic considerations, the absolute configuration of compound 6 was assigned as 6R, 7R, 8R, 10S, 12S, and 13S. Compound 6 was named 18-O-acetyltanzawaic acid R.
Steckwaic acid I (7) was obtained as a white amorphous powder, and the molecular formula was assigned as C19H28O4 by analysis of the HRESIMS data. A detailed compari-  Compound 6 was isolated as a white amorphous powder and assigned the molecular formula C20H28O5 by negative HRESIMS data. A detailed comparison of the NMR spectral data (Table 2) with the known compound tanzawaic acid R [17] suggested that they were very similar. However, signals for a carboxyl carbon at δC 170.4 and a methyl group at δC/δH 20.7/2.00 related to an acetoxyl group were observed in the NMR spectra of 6. HMBC correlations from H2-18 to C-9, C-11, and C-19 as well as H3-20 to C-19 placed the acetoxyl group at C-18 ( Figure 2). Large coupling constants between H-2 and H-3, as well as between H-4 and H-5 (J = 15.3 Hz), indicated the E-geometry of two double bonds. NOESY correlations from H3-17 to H-5, H-7, and H2-18, as well as from H2-18 to H-11β, suggested the same orientation of these protons, while correlations from H-12 to H-8 and H-10 as well as from H-13 to H-11α placed these groups on the opposite face. From the ECD data and biogenetic considerations, the absolute configuration of compound 6 was assigned as 6R, 7R, 8R, 10S, 12S, and 13S. Compound 6 was named 18-O-acetyltanzawaic acid R.
Steckwaic acid I (7) was obtained as a white amorphous powder, and the molecular formula was assigned as C19H28O4 by analysis of the HRESIMS data. A detailed comparison of the NMR spectral data between 7 (Table 3) and tanzawaic acid S (10) [17] suggested Compound 6 was isolated as a white amorphous powder and assigned the molecular formula C 20 H 28 O 5 by negative HRESIMS data. A detailed comparison of the NMR spectral data (Table 2) with the known compound tanzawaic acid R [17] suggested that they were very similar. However, signals for a carboxyl carbon at δ C 170.4 and a methyl group at δ C /δ H 20.7/2.00 related to an acetoxyl group were observed in the NMR spectra of 6. HMBC correlations from H 2 -18 to C-9, C-11, and C-19 as well as H 3 -20 to C-19 placed the acetoxyl group at C-18 ( Figure 2). Large coupling constants between H-2 and H-3, as well as between , indicated the E-geometry of two double bonds. NOESY correlations from H 3 -17 to H-5, H-7, and H 2 -18, as well as from H 2 -18 to H-11β, suggested the same orientation of these protons, while correlations from H-12 to H-8 and H-10 as well as from H-13 to H-11α placed these groups on the opposite face. From the ECD data and biogenetic considerations, the absolute configuration of compound 6 was assigned as 6R, 7R, 8R, 10S, 12S, and 13S. Compound 6 was named 18-O-acetyltanzawaic acid R.
Steckwaic acid I (7) was obtained as a white amorphous powder, and the molecular formula was assigned as C 19 H 28 O 4 by analysis of the HRESIMS data. A detailed comparison of the NMR spectral data between 7 (Table 3) and tanzawaic acid S (10) [17] suggested that they were very similar, except the coupling constant between H-4 and H-5 (J = 11.1 Hz) in 7 was much smaller than that of 10 (J 4,5 = 15.3 Hz), indicating the geometry of the double bond at C-4 changed from E in 10 to Z in 7. The planar structure was further determined by COSY and HMBC correlations (Figure 2), and the relative configuration was confirmed by the NOESY spectrum. The NOESY correlations from H-5 to H-7 and H 3 -17 showed these groups to be on the same side, whereas correlations from H- 6 to H-8, H-12, and H-13, as well as from H-10 to H-8 and H-12, indicated the opposite side of these protons. The absolute configuration of 7 was confirmed by comparing the ECD spectrum with those of known compounds 9 and 10. The same positive Cotton effects around 265 nm demonstrated consistent absolute configurations with those of 6R, 7R, 8R, 10S, 12S, and 13S.  (15.4,11.1) 144.5,CH 7.12,dd (15.3,11.0) 4 129.6,CH 6.22,td (11.1,5.2) 129. 6,CH 6.28,dd (15.3,11. Compound 8 was also obtained as a white amorphous powder with the molecular formula C 19 H 28 O 4 as measured by HRESIMS. Analysis of 1 H and 13 C NMR data (Table 3) showed similarities to those reported for tanzawaic acid S (10) that were measured in methanol-d 4 [17] and our isolates that were measured in DMSO-d 6 (Experimental section). The primary differences between 8 and 10 were the signals of an oxygenated methine at δ C /δ H 80.3/3.32 (CH-13) in 8 replaced by signals at δ C /δ H 75.3/3.37 (CH-13) in 10, revealing the configuration at C-13 had been changed. This deduction was further supported by the relevant COSY and HMBC correlations shown in Figure 2, as well as NOESY data shown in Figure 3. NOE correlations from H-5 to H-7 and H 3 -17, from H-7 to H-13, and from H 2 -18 to H-11β suggested they were on one side, whereas correlations from H-6 to H-8 and H-12 and from H-13-OCH 3 to H-10 and H-11α placed them on another face. By comparing the ECD spectrum with that of compounds 9 and 10, the absolute configuration of 8 was assigned as 6R, 7R, 8R, 10S, 12S, and 13R. Compound 8 was named 13R-tanzawaic acid S.
In addition to the new compounds 1−8, two structurally related known analogues 9 and 10 were also isolated, and their structures were identified as tanzawaic acid H (9) and tanzawaic acid S (10) based on the comparison of NMR data, optical rotations, and ECD spectra with those described in literature reports [16,17].
The absolute configurations of major chiral centers for compounds 1−10 are consistent except for position C-13. In comparison to 13S methoxy-substituent (compounds 5, 7, and 10), the resonance of C-13 in the 13R isomer (compound 8) shifted~5 ppm downfield in the 13 C NMR spectra. An analogous carbon (4.4 ppm) was observed for the analogous hydroxysubstituent when comparing compound 6 with compound 9. Besides, for the compounds with the same absolute configuration at C-13 (5, 6, 7, and 10), the methoxy-substituent (5, 7, and 10) could shift downfield approximately 10 ppm in the 13 C NMR spectra compared to that of hydroxy-substituent (6).
Although some tanzawaic acid derivatives were tested for their cytotoxic [16] and lipidlowering [17] activities, a few of them exhibited significant activities. Compounds 1-10 were assayed for their antibacterial activities against one human and nine aquatic pathogenic bacteria as well as seven plant-pathogenic fungi. Compound 8 showed moderate activity against the human pathogenic bacterium Escherichia coli with an MIC value of 8 µg/mL (the MIC value of the positive control chloramphenicol was 1 µg/mL), while compound 10 exhibited inhibitory activity against the aquatic pathogenic bacterium Edwardsiella tarda with an MIC value of 16 µg/mL (the MIC value of the positive control chloramphenicol was 2 µg/mL) ( Table 4). The results suggested that the absolute configuration of C-13 influenced the antibacterial activities of different bacteria (8 vs. 10) as supported by the fact that compound 8 with a 13R configuration showed stronger activity against E. coli but weaker activity against E. tarda, while compound 10 with a 10S configuration showed stronger activity against E. tarda but no activity against E. coli. The geometry of the double bond at C-4 also affected the activity (7 vs. 10), revealed by the fact that the E-geometry (10) exhibited stronger activity against E. tarda while compound 7 with Z-geometry at C-4 did not show any activity. Other compounds did not exhibit antimicrobial activities (MIC > 64 µg/mL).

General Experimental Procedures
General experimental procedures were the same as previously reported [19,20].

Fungal Material
The fungal strain Penicillium steckii AS-324 was obtained from fresh tissues of Acanthogorgiidae sp., which were collected from Magellan seamount. Taxonomic identification of the fungus was accomplished by comparing its ITS region sequence to that of Penicillium steckii (MT582790.1), which showed 99.64% similarity. The sequence data of the fungus AS-324 were submitted and deposited in GenBank with the accession no. OK605032. The fungal strain is preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (IOCAS, Qingdao, China).