Absolute Configurations of 14,15-Hydroxylated Prenylxanthones from a Marine-Derived Aspergillus sp. Fungus by Chiroptical Methods

Determination of the absolute configrations for natural products is one of the most important and challenging tasks, especially when the molecules display high conformational flexibility. In this paper, eight new prenylxanthones, aspergixanthones A-H (1–8), and one known analogue (9), were isolated from the marine-derived fungus Aspergillus sp. ZA-01. The absolute configurations of C-14 and C-15 in 1–8 were difficult to be assigned due to the high conformational flexibility of the chains. To solve this problem, the experimental ECD, ORD, and VCD spectra of 1 were combined for analysis with the corresponding theoretical predictions for its different diastereomers. This study suggested that a concerted application of more than one chiroptical methods could be used as a preferable approach for the stereochemical characterizations of flexible molecules. Compounds 1–9 were evaluated for their cytotoxic and antibacterial activities. Among them, 6 showed cytotoxicity against the A-549 cell line with the IC50 value of 1.1 μM, and 7 exhibited antibacterial activity against Micrococcus lysodeikticus with the MIC value of 0.78 μg/mL.

SCiEnTifiC RepoRts | (2018) 8:10621 | DOI:10.1038/s41598-018-28996- 5 Aspergixanthone B (2) was also obtained as a yellow powder. Its molecular formula was detetermined as C 26 H 30 O 8 by positive HRESIMS, revealing the loss of a -COCH 3 group compared with that of 1. The NMR data (Tables 1 and 2) revealed that 2 had the same structural features as those of 1, except for the absence of an acetoxy at C-25 in 2. This observation was further demonstrated by the 1 H-1 H COSY cross-peaks of OH-25/H-25/H-20. Compound 2 was thus identified as the 25-deacetylation derivative of 1.
Aspergixanthones C and D (3 and 4) were also isolated as yellow powder with the molecular formulas of C 27 H 30 O 9 and C 25 H 28 O 8 by positive HRESIMS, respectively. The 1D and 2D NMR data of 3 and 4 (Tables 1 and 2) revealed their similar prenylxanthone nucleus to those of 1 and 2. Particularly, the NMR data showed the presence of a C-25 acetoxy group in 3 and a C-25 hydroxy group in 4. Detailed analysis and comparison of the NMR and MS data of 3 and 4 with those of 1 and 2 revealed that the methoxy at C-14 in 1 and 2 was absent in 3 and 4, which was verified by the each HMBC correlations from H-14 to C-3, C-4 and C-15 in 3 and 4.
Aspergixanthones E and F (5 and 6) displayed quasi-molecular ions at m/z 577.2048 and 535.1539 [M + Na] + in the positive HRESIMS, corresponding to the molecular formulas of C 30 H 34 O 10 and C 28 H 32 O 9 , respectively. Compounds 5 and 6 were also prenylxanthone analogues by the comparison of the strikingly similar NMR data of 5 and 6 (Tables 2 and 3) with those of 1 and 2, with the appearance of the additional acetoxy groups in 5 and 6. The additional acetoxys laid within their respective side chains at C-15, demonstrated by the detailed analysis of 1 H-1 H COSY and HMBC spectra of 5 and 6, respectively. Aspergixanthones G and H (7 and 8) had the molecular formulas of C 27 H 28 O 8 and C 25 H 26 O 7 , respectively. The NMR data (Tables 2 and 3) of 7 and 8 were similar to those of 3 and 4, except that the presence of disubstituted double bond signals in 7 and 8 instead of the methyls in 3 and 4. Comprehensive analysis of 2D NMR data of 7 and 8 supported that the C-17 methyls in 3 and 4 were oxidized to form the corresponding olefinic bonds in 7 and 8, respectively.
The relative configurations of C-20 and C-25 of 1-8 were assigned by NOESY experiments and comparison of the NMR data with those of reported prenylxanthone derivatives [3][4][5][6] . In the NOESY spectra of 1-3 and 5-8, the correlation between H-25 and CH 2 -22/CH 3 -23 was observed, suggesting these protons should be on the same face of the molecule. And, the 1 H NMR data about δ H 2.71 for H-20 suggested a trans-diaxal relationship of hydroxy and isopropenyl in 1-3 and 5-8, which was consistent with the corresponding configuration of tajixanthone hydrate [4][5][6] . Whereas, the NOESY correlation between OH-25 and CH 3 -23 was unobserved and the 1 H NMR data about δ H 2.51 for H-20 indicated the cis-related relationship of H-20 and H-25 (twist-chair conformation) in 4, which was close to literature values for known compound epitajixanthone hydrate (δ H 2.55) 3  In recent years, three most frequently used chiroptical methods, namely electronic circular dichroism (ECD), optical rotatory dispersion (ORD), and vibrational circular dichroism (VCD) have proven to be useful means for the stereochemical characterizations of natural products [17][18][19] . However, none of these chiroptical techniques was capable of dominating stereochemical characterizations, as they each had their respective limitations for the different structures 20 . ECD had one or two orders of magnitude higher sensitivity, but the stereogenic centers should be close to UV-Vis chromophores 21 . ORD has been popularly used in recent research, yet, it was hard to explain the spectra 21 . VCD, which require no chromophores in the UV-Vis region and have a larger scope than ECD, was limited by the sample quantity 21 . Thus, the use of more than one chiroptical techniques could provide more reliable results for complex products, especially for some flexible compounds 20,21 . In this paper, a combined analysis of ECD, ORD, and VCD properties was applied to elucidate the absolute configuration of conformationally flexible 1.   Boltzmann-weighted ECD spectra of the four diastereomers were generated using the SpecDis 1.6 soft with a standard deviation of σ 0.2 eV. As shown in Fig. 3, all of the predicted ECD curves for the four diastereomers matched well with the measured ECD spectrum of 1, suggesting that the absolute configurations of C-20 and C-25 in 1 were assigned as 20S, 25R.
Recently, VCD approach has become robust and reliable alternative for the stereochemical characterizations of natural products, especially in conditions not accessible to the other methods [22][23][24] . The experimental IR and VCD spectra of 1 (12.0 mg) were measured in 120 μL of DMSO-d 6 using a BioTools dual PEM ChiralIR-2X spectrophotometer. The IR and VCD frequencies of (14R, 15R, 20S, 25R)-1 and (14R, 15S, 20S, 25R)-1 were calculated at the gas-phase PBEPBE/6-311 + G(d)//PBEPBE/6-311 + G(d) level to compare the experimental IR and VCD data of 1. All most of the calculated VCD signals of (14R, 15R, 20S, 25R)-1 had agreements with the experimental VCD signals of 1, while the signals of 4, 8 and 9 in the calculated VCD spectrum of (14R, 15S, 20S, 25R)-1 had disagreements with the corresponding signals in the experimental VCD spectrum of 1 (Fig. 5), suggesting that the structure of (14R, 15R,20S,25R)-1 was closer to the real structure of 1. In order to further verify the absolute configuration of 1, the dimolybdenum tetraacetate [Mo 2 (AcO) 4 ] induced circular dichroism (ICD) procedure (Snatzke′s method) was used. The negative ICD Cotton effects around 300 and 400 nm of 1 (Fig. 6) gave the newman form of Mo-complexes of 1. It was found that a counterclockwise rotation, suggesting the R configuration for C-15 in 1. Therefore, on the basis of the above ECD, ORD, VCD, and Snatzke′s results, the absolute configuration of 1 could be defined as 14R, 15R, 20S, 25R, unambiguously.
It was showed that the ECD spectra of 2-8 closely matched that of 1 ( Figures S1 and S2), suggesting the 20S, 25R absolute configuration for 2-3 and 5-8, and 20R, 25R for 4. Due to the sample quantity limitation of 2-8, it was difficult to elucidate the absolute configurations of C-14 and C-15 in 2-8 by VCD method, directly. Their absolute configurations could be tentatively assigned on the basis of a shared biogenesis with the co-isolated 1, whose absolute configuration had been unambiguously established firstly. The absolute configurations could be proposed as 14R, 15S, 20S, 25R for 2, 3, 5 and 6, 14R, 15S, 20R, 25R for 4, and 14R, 15R, 20S, 25R for 7 and 8.
Xanthones, which were isolated from many different species within fungi, bacteria, and higher plants are widespread classes of typically polysubstituted dibenzo-γ-pyrone derivatives 25,26 . Among them, prenylxanthones  (1-9) represent the family of naturally occurring xanthones with C-4 terpenoid-derived side chain 25 . In the previous literature, twenty prenylxanthones have been mainly obtained from the genus Aspergillus/Emericella fungi, such as 14-hydroxyltajixanthone hydrate 3 , emerixanthones A-D 4 , ruguloxanthones A-C 27 , and so on. The configurations of C-14 and C-15 in prenylxanthone derivatives were challenging to be assigned due to the high conformational flexibility of the terpenoid-derived side chains. It was un-accommodated to determine the relative configuration of C-14 and C-15 by comparison of the coupling constants (J 14,15 ) as the free rotation of the flexible side chain 5 . Also, it was problem to define the absolute configuration of C-15 by comparison of the optical rotatory 27 . This work demonstrated that using multiple chiroptical methods in combination with DFT calculations allowed one to determine absolute configurations with high confidence for chiral natural products, which possessed rotatable bonds. Compounds 1-9 were subjected to test their cytotoxic activities by MTT method against human breast cancer (MDA-MB-231 and MCF-7), human gastric cancer (MGC-803), cervical cancer (HeLa), and human lung epithelial carcinoma (A-549) cell lines. Compound 1 displayed selective cytotoxicity against the A-549 cell line with the IC 50 value of 1.8 μM. Compounds 3 and 6 showed broad-spectrum cytotoxicities against five tumor cell lines with the IC 50 values ranging from 1.1 to 9.8 μM (Table 4). However, the other compounds (2,4,5,7,8,9) exhibited very low cytotoxicity to any of the above cell lines (IC 50 > 10.0 μM).
Compounds 1-9 were further tested their antibacterial activities against a panel of pathogenic bacteria, including Micrococcus lysodeikticus, Bacillus anthraci, Salmonella typhi, and Enterobacter aerogenes. Only 7 and 8 showed antibacterial activity against M. lysodeikticus,B. anthraci,S. typhi,and E. aerogenes,with MIC values of 0.78,12.5,6.13 and 6.13 μg/mL for 7,and 6.13,12.5,6.13 and 6.13 μg/mL for 8, respectively. These data indicated that the antibacterial activities may be due to the double bonds between C-16 and C-17 in 7 and 8. In addition, ciprofloxacin showed antibacterial activity against M. lysodeikticus, B. anthraci, S. typhi, and E. aerogenes, with MIC values of 0.19, 1.56, 3.13, 1.56 μg/mL, respectively.  In summary, three chiroptical methods, including ECD, ORD and VCD, combined with quantum theory calculation were carried out to elucidate the absolute configuration of the prenylxanthone 1, which was difficult to be determined by single method due to high flexibility of the molecule. The interesting chemical structures and  potent biological activities of these prenylxanthones (1-9) may encourage further investigations on this cluster of metabolites for drug discovery.

Methods
General Experimental Procedures. Optical rotatory dispersions were acquired using a JASCO P-2000 spectrometer. Optical rotations were obtained on an Optical Activity AA-55 series polarimeter. UV data were performed on a Perkin-Elmer model 241 spectrophotometer in MeOH. Electronic circular dichroism spectra were measured using a JASCO J-715 circular dichroism spectrometer. IR spectra were determined using KBr pellets with a Nicolet NEXUS 470 spectrophotometer. Vibrational circular dichroism spectra were taken on a BioTools ChiralIR-2X spectrophotometer. 1D and 2D NMR data (600 MHz for 1 H and 150 MHz for 13 C) were acquired on Bruker Avance-III 600 MHz NMR spectrometer with TMS as an internal standard. High-resolution mass data were obtained from a Thermo Scientific LTQ Orbitrap XL spectrometer. HPLC analysis and semi-preparation was performed on a Shimadzu LC-20AT system with a SPD-M20A photodiode array detector, using a Waters RP-18 (XBridge OBD, 5 μm, 10 × 250 mm) and a Waters normal phase (Viridis TM Silica 2-Ethylpyridine, 5 μm, 10 × 250 mm) columns. Column chromatography was performed on Silica gel 200-300 mesh (Qingdao Marine Chemical Factory), and Sephadex LH-20 Pharmacia Fine Chemical Co.,Ltd.,Sweden). Extraction and Purification. The fermentation of the fungus Aspergillus sp. ZA-01 was carried out using  Table 4. Cytotoxicity of compounds 1-9.