Two Antimicrobial Heterodimeric Tetrahydroxanthones with a 7,7′-Linkage from Mangrove Endophytic Fungus Aspergillus flavus QQYZ

Mangrove endophytic fungi represent significant and sustainable sources of novel metabolites with unique structures and excellent biological activities, attracting extensive chemical investigations. In this research, two novel heterodimeric tetrahydroxanthones, aflaxanthones A (1) and B (2), dimerized via an unprecedented 7,7′-linkage, a sp3-sp3 dimeric manner, were isolated from the mangrove endophytic fungus Aspergillus flavus QQYZ. Their structures were elucidated through high resolution electrospray ionization mass spectroscopy (HRESIMS) and nuclear magnetic resonance (NMR) spectroscopy, the absolute configurations of them were determined by a single-crystal X-ray diffraction combined with calculated electronic circular dichroism (ECD) spectra and a 1D potential energy scan. These compounds were evaluated for antifungal activities in vitro and exhibited broad-spectrum and potential antifungal activities against several pathogenic fungi with minimum inhibitory concentration (MIC) values in the range of 3.13–50 μM. They also performed moderate antibacterial activities against several bacteria with MIC values in the range of 12.5–25 μM. This research enriched the resources of lead compounds and templates for marine-derived antimicrobial drugs.

Previously, a series of novel bioactive metabolites were characterized from mangrove endophytic fungi in the South China Sea, in our group [24][25][26][27][28][29]; during ongoing research, a chemical investigation was performed on an endophytic fungus, Aspergillus flavus QQYZ isolated from a fresh blade of the mangrove plant Kandelia candel, collected from Huizhou in Guangdong province. Two new heterodimeric tetrahydroxanthones, aflaxanthones A (1) and B (2), which were dimerized via an unprecedented 7,7′-linkage, a nonbiaryl dimeric manner, were obtained from the culture broth of the fungus (Figure 1). It represents the first report of xanthone dimers formed by a non-aromatic single bond connection. In the antimicrobial activities assays, these new compounds have exhibited broad-spectrum and pronounced antifungal and antibacterial activities. Herein, we report the isolation, structure elucidation, and antimicrobial activities of the two compounds.

Results
After 28 days of cultivation on rice solid medium, the EtOAc extract of fungus Aspergillus flavus QQYZ was fractionated with column chromatography, using silica gel and Sephadex LH-20, followed by chiral high performance liquid chromatography (HPLC) for the two novel compounds from a mixed component.

Results
After 28 days of cultivation on rice solid medium, the EtOAc extract of fungus Aspergillus flavus QQYZ was fractionated with column chromatography, using silica gel and Sephadex LH-20, followed by chiral high performance liquid chromatography (HPLC) for the two novel compounds from a mixed component.  3 -12 ). Notably, unlike xanthone dimers that are typically dimerized through a biaryl bond, compound 1 had four aromatic protons instead of two [2,7,23]. Moreover, the hydroxymethyl at δ H 4.51 ppm and methyl at δ H 2.25 ppm, combined with the molecular formula, can exclude the linkage of an ether bond compared with asperdichrome and 5epi-asperdichrome [22,30]. Thus, there might be a new linkage of dimerization. Combined with the HSQC, there were 18 sp 2 -hybridized carbons and 12 sp 3 carbons in the 13 C NMR spectrum, including two ketone carbonyls (δ C 188. 6  , and 37.1), two methylenes (δ C 29.9 and 28.0), one hydroxymethyl (δ C 64.1), and three methyl carbons (δ C 26.1, 22.5, and 20.1). The NMR signals of 1 appeared duplication but not overlapping; moreover, C-11 (δ H 4.51, δ C 64.1) and C-11 (δ H 2.25, δ C 22.5) exhibited in a single-handed form, meaning a heterodimeric skeleton with a minute difference of 3-methyl and 3-hydroxymethyl between the planar structure of the two units. The heteronuclear multiple bond correlation (HMBC) from H-2 to C-1, C-4, C-9, and C11, from H-4 to C-2, C-4a, C-9, and C-11, from H-11 to C-2, C-3, and C-4, implied the presence of a 1,2,4,6-tetrasubstituted benzene system ( Figure 2). The 1 H-1 H homonuclear chemical shift correlation spectroscopy (COSY) cross-peak of H-5/H-6 and H-6/H-7, along with the HMBC correlations from H-5 to C-6, C-7, C-8a, C-10a, and C-12, from H-12 to C-5, C-8a, and C-10a, from H-6 to C-7, C-8, and C-7 illustrated a 1,2,3,4,6-pentasubstituted cyclohexene moiety. The aforementioned ketone groups, benzene and cyclohexene rings in two units, accounted for 14 degrees of unsaturation, the remaining 2 degrees of unsaturation in conjunction with a further HMBC correlation from H-5 to C-9 and the chemical shift of C-4a and C-10a indicated the linkage of the two moieties via C8a-C9/C8a'-C9 and C4a-O-C10a/C4a -O-C10a to form a tetrahydroxanthone scaffold. As for the connection of two fragments, both benzene ring fragments had two proton signals, indicating that the dimerization mode was different from the common mode of a biaryl bond [7,14,23]. However, the 7/7 -methines and the HMBC correlations from H-6 to C-7 , from H-6 to C-7, indicated the two units dimerized via a unique 7,7 -bond that may have been discovered in xanthone dimers for the first time, which was later proved by the X-ray diffraction experiment.  The relative configuration of 1 can be revealed by the nuclear overhauser effect spectroscopy (NOESY) spectrogram ( Figure 2), as the key correlations of H-5 with H3-12 suggested that H-5 and H3-12 positioned on the same face and H-7 on the other face. The NOESY correlation of H-5′ with H-7′ implied H-5′ and H-7′ had the same orientation and the H3-12′ was in the opposite direction. The above NOESY correlations showed different chirality of the two units at aliphatic parts of C-5/C-5′, C-7/C-7′, and C-10a/C-10a'. Fortunately, the single crystals of 1 were crystallized slowly in the mixture of methanol and dichloromethane with a same volume ratio. Thus, the absolute configuration of 1 was confirmed by the following X-ray diffraction analysis using the Cu Kα radiation with a flack parameter of 0.02(13) ( Figure 3). Meanwhile, to further verify the stereo structure, ECD calculations were carried out by the time-dependent density functional theory (TDDFT) approach at B3LYP/6−311+g (d,p) level [31], using the single-crystal stereochemical structure as input. The results of the theoretical ECD spectra basically showed a consistent Cotton effect with the experimental curve in acetonitrile ( Figure 4). Consequently, the absolute configuration of compound 1 was determined as 5S,7R,10aR,5′S,7′S,10a'S. The results of a single crystal diffraction analysis and theoretical ECD spectra strongly proved that this was the first discovery of a xanthone dimer connected by a 7-7′ linkage of a nonbiaryl bond.  The relative configuration of 1 can be revealed by the nuclear overhauser effect spectroscopy (NOESY) spectrogram (Figure 2), as the key correlations of H-5 with H 3 -12 suggested that H-5 and H 3 -12 positioned on the same face and H-7 on the other face. The NOESY correlation of H-5 with H-7 implied H-5 and H-7 had the same orientation and the H 3 -12 was in the opposite direction. The above NOESY correlations showed different chirality of the two units at aliphatic parts of C-5/C-5 , C-7/C-7 , and C-10a/C-10a'. Fortunately, the single crystals of 1 were crystallized slowly in the mixture of methanol and dichloromethane with a same volume ratio. Thus, the absolute configuration of 1 was confirmed by the following X-ray diffraction analysis using the Cu Kα radiation with a flack parameter of 0.02 (13) (Figure 3). Meanwhile, to further verify the stereo structure, ECD calculations were carried out by the time-dependent density functional theory (TDDFT) approach at B3LYP/6-311+g (d,p) level [31], using the single-crystal stereochemical structure as input. The results of the theoretical ECD spectra basically showed a consistent Cotton effect with the experimental curve in acetonitrile ( Figure 4). Consequently, the absolute configuration of compound 1 was determined as 5S,7R,10aR,5 S,7 S,10a'S. The results of a single crystal diffraction analysis and theoretical ECD spectra strongly proved that this was the first discovery of a xanthone dimer connected by a 7-7 linkage of a non-biaryl bond.  The relative configuration of 1 can be revealed by the nuclear overhauser effect spectroscopy (NOESY) spectrogram (Figure 2), as the key correlations of H-5 with H3-12 suggested that H-5 and H3-12 positioned on the same face and H-7 on the other face. The NOESY correlation of H-5′ with H-7′ implied H-5′ and H-7′ had the same orientation and the H3-12′ was in the opposite direction. The above NOESY correlations showed different chirality of the two units at aliphatic parts of C-5/C-5′, C-7/C-7′, and C-10a/C-10a'. Fortunately, the single crystals of 1 were crystallized slowly in the mixture of methanol and dichloromethane with a same volume ratio. Thus, the absolute configuration of 1 was confirmed by the following X-ray diffraction analysis using the Cu Kα radiation with a flack parameter of 0.02 (13) (Figure 3). Meanwhile, to further verify the stereo structure, ECD calculations were carried out by the time-dependent density functional theory (TDDFT) approach at B3LYP/6−311+g (d,p) level [31], using the single-crystal stereochemical structure as input. The results of the theoretical ECD spectra basically showed a consistent Cotton effect with the experimental curve in acetonitrile ( Figure 4). Consequently, the absolute configuration of compound 1 was determined as 5S,7R,10aR,5′S,7′S,10a'S. The results of a single crystal diffraction analysis and theoretical ECD spectra strongly proved that this was the first discovery of a xanthone dimer connected by a 7-7′ linkage of a nonbiaryl bond.   Aflaxanthone B (2) was also acquired as a yellow powder, sharing the same molecular formula of C30H30O11 as 1, established by HRESIMS data at m/z 589.1676 ([M + Na] + , calcd for C30H30O11Na, 589.1680). Since showing the same UV data as 1, it suggested that 2 was also a tetrahydroxanthone derivative. To deduce the structure of 2, the NMR experiments were implemented at room temperature, 298 K. However, confusingly, in the 1 H NMR spectrum of 2, the aliphatic protons of H-5/5′, H2-6/6′ and H-7/7′ were unable to be observed contrasted with 1 ( Table 1). The same phenomenon appeared in the 13 C NMR spectrum, as the carbon signals of C-5/5′, C-6/6′, C-7/7′, C-8/8′, and C-8a/8a' were absent, the signals of the connection moiety between the two units disappeared, then the lowtemperature 1 H NMR experiments were preliminarily conducted under 273 and 243 K to clarify the integral structure of 2 [31][32][33]. Broad peaks at 273 K and obvious resonance peaks at 243 K emerged ( Figures S12 and S13), signifying the NMR experiments at 243 K can be feasible; meanwhile, signals in the low field of 13 C NMR were separating at 243 K, indicating rotational isomers existed in 2 [34]. Further comprehensive analysis of the new set of NMR spectra (Table 1) experiments performed at 243 K with clear carbon peaks and correlations of the 2D NMR spectrum, especially a striking key 1 H-1 H COSY cross-peak of H-7/H-7′ (Figure 2), showed the same planar structure as 1 dimerized with a 7,7′-linkage.
The key NOESY correlations of H-5 with H-12 and H-5′ with H-12′ and the deficiency of interactions of H-7 with H-5 and H-7′ with H-5′ evinced the same central chirality of the two units ( Figure 2). The single crystal of 2 was obtained under the same conditions as 1, and the subsequent X-ray diffraction experiment confirmed the absolute configuration of 2 to be 5S,7R,10aR,5′S,7′R,10a'R (Figure 3), giving a flack parameter of −0.08 (5). Owing to the asymmetry by the 3-hydroxymethyl and the 3′-methyl, but with the same appearing ratio, a disorder existed in the X-ray diffraction [35]. Then the analogy was made between the theoretical and experimental ECD spectra by the same approach as 1, and consistent Cotton effects of experimental and calculated curves were observed ( Figure 4). Ultimately, the absolute configuration of 2 was unambiguously elucidated as 5S,7R,10aR,5′S,7′R,10a'R; it was also proved to process the unique 7-7′-linkage.
A portion of the NMR signal around the chiral axis could broaden or disappear in some axial chiral compounds [32,34,36]; in order to verify the presence of the axial chirality in 2, a 1D potential energy scan (PES) was conducted on the dihedral angle C8-C7-C7′-C8′ by modredundant optimization using the DFT method at the B3LYP/6−31g (d,p) level in Gaussian 09 [31] to calculate the rotational energy barrier around the C7-C7′ bond (Figure 5) [19]. The relative Gibbs energy barriers at each transition state (TS) for the M/P conversion were 21.35 kcal/mol (TS2-1) and 14.41 kcal/mol (TS2-2), indicating the coalescence of the M/P isomer in 2 at room temperature [31,37]. On account of the interconversion in 2, the absence of the NMR signals around the C7-C7′ bond at 298 K occurred, and the 13 C Aflaxanthone B (2) was also acquired as a yellow powder, sharing the same molecular formula of C 30  Since showing the same UV data as 1, it suggested that 2 was also a tetrahydroxanthone derivative. To deduce the structure of 2, the NMR experiments were implemented at room temperature, 298 K. However, confusingly, in the 1 H NMR spectrum of 2, the aliphatic protons of H-5/5 , H 2 -6/6 and H-7/7 were unable to be observed contrasted with 1 ( Table 1). The same phenomenon appeared in the 13 C NMR spectrum, as the carbon signals of C-5/5 , C-6/6 , C-7/7 , C-8/8 , and C-8a/8a' were absent, the signals of the connection moiety between the two units disappeared, then the low-temperature 1 H NMR experiments were preliminarily conducted under 273 and 243 K to clarify the integral structure of 2 [31][32][33]. Broad peaks at 273 K and obvious resonance peaks at 243 K emerged (Figures S12 and S13), signifying the NMR experiments at 243 K can be feasible; meanwhile, signals in the low field of 13 C NMR were separating at 243 K, indicating rotational isomers existed in 2 [34]. Further comprehensive analysis of the new set of NMR spectra (Table 1) experiments performed at 243 K with clear carbon peaks and correlations of the 2D NMR spectrum, especially a striking key 1 H-1 H COSY cross-peak of H-7/H-7 (Figure 2), showed the same planar structure as 1 dimerized with a 7,7 -linkage.
The key NOESY correlations of H-5 with H-12 and H-5 with H-12 and the deficiency of interactions of H-7 with H-5 and H-7 with H-5 evinced the same central chirality of the two units ( Figure 2). The single crystal of 2 was obtained under the same conditions as 1, and the subsequent X-ray diffraction experiment confirmed the absolute configuration of 2 to be 5S,7R,10aR,5 S,7 R,10a'R (Figure 3), giving a flack parameter of −0.08 (5). Owing to the asymmetry by the 3-hydroxymethyl and the 3 -methyl, but with the same appearing ratio, a disorder existed in the X-ray diffraction [35]. Then the analogy was made between the theoretical and experimental ECD spectra by the same approach as 1, and consistent Cotton effects of experimental and calculated curves were observed ( Figure 4). Ultimately, the absolute configuration of 2 was unambiguously elucidated as 5S,7R,10aR,5 S,7 R,10a'R; it was also proved to process the unique 7-7 -linkage.
A portion of the NMR signal around the chiral axis could broaden or disappear in some axial chiral compounds [32,34,36]; in order to verify the presence of the axial chirality in 2, a 1D potential energy scan (PES) was conducted on the dihedral angle C8-C7-C7 -C8 by modredundant optimization using the DFT method at the B3LYP/6-31 g (d,p) level in Gaussian 09 [31] to calculate the rotational energy barrier around the C7-C7 bond ( Figure 5) [19]. The relative Gibbs energy barriers at each transition state (TS) for the M/P conversion were 21.35 kcal/mol (TS2-1) and 14.41 kcal/mol (TS2-2), indicating the coalescence of the M/P isomer in 2 at room temperature [31,37]. On account of the interconversion in 2, the absence of the NMR signals around the C7-C7 bond at 298 K occurred, and the 13 C NMR spectrum showed separated signals of the chromone moieties at 243 K simultaneously, suggesting the 8-OH and 8 -OH cannot impose sufficient spatial hindrances to interrupt the free rotation of the two units [36,38]. As an additional supplement, we also calculated the Gibbs energy barrier of 1; the results were 6.67 kcal/mol (TS1-1) and 12.90 kcal/mol (TS1-2), which meant the two units of 1 could rotate freely and 1 could be developed as a single compound [32], and a complete set of NMR signals of 1 at room temperature could be observed as proof. Hence, the two compounds can be determined as optically pure monomers [16,20]. Moreover, the results above could mean that there might also be axial chirality around the sp 3 -sp 3 hybrid carbon-carbon bond. If the space occupations of the groups on both sides of the axis are large enough and the signals in NMR change, it may be necessary to verify the existence of axial chirality. NMR spectrum showed separated signals of the chromone moieties at 243 K simultaneously, suggesting the 8-OH and 8′-OH cannot impose sufficient spatial hindrances to interrupt the free rotation of the two units [36,38]. As an additional supplement, we also calculated the Gibbs energy barrier of 1; the results were 6.67 kcal/mol (TS1-1) and 12.90 kcal/mol (TS1-2), which meant the two units of 1 could rotate freely and 1 could be developed as a single compound [32], and a complete set of NMR signals of 1 at room temperature could be observed as proof. Hence, the two compounds can be determined as optically pure monomers [16,20]. Moreover, the results above could mean that there might also be axial chirality around the sp 3 -sp 3 hybrid carbon-carbon bond. If the space occupations of the groups on both sides of the axis are large enough and the signals in NMR change, it may be necessary to verify the existence of axial chirality. Compounds 1 and 2 were evaluated for antifungal activities against Candida albicans and four agricultural plant pathogenic fungi-Fusarium oxysporum, Penicillium italicum, Collettrichum musae, and Colletotrichum gloeosporioides; 1 showed promising inhibitory activity against C. gloeosporioides, for 3.13 μM, while the MIC of the positive control ketoconazole was 0.1 μM, and moderate activity against F. oxysporum and C. albicans with MIC was 12.5 μM; 2 exhibited moderate activity against F. oxysporum and C. musae with MIC 12.5 μM, respectively ( Table 2). As for antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Bacillus subtilis, compound 1 possessed moderate inhibitory activity against MRSA with a MIC value 12.5 μM, and the two compounds had moderate inhibitory effects on B. subtilis with MIC 25 μM compared to the positive control ampicillin with MIC 0.39 and 0.39 μM against MRSA and B. subtilis, respectively ( Table 2). The experiments show that the compounds could be used as spectral antifungal and antibacterial drug precursors for more in-depth research.  Compounds 1 and 2 were evaluated for antifungal activities against Candida albicans and four agricultural plant pathogenic fungi-Fusarium oxysporum, Penicillium italicum, Collettrichum musae, and Colletotrichum gloeosporioides; 1 showed promising inhibitory activity against C. gloeosporioides, for 3.13 µM, while the MIC of the positive control ketoconazole was 0.1 µM, and moderate activity against F. oxysporum and C. albicans with MIC was 12.5 µM; 2 exhibited moderate activity against F. oxysporum and C. musae with MIC 12.5 µM, respectively ( Table 2). As for antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Bacillus subtilis, compound 1 possessed moderate inhibitory activity against MRSA with a MIC value 12.5 µM, and the two compounds had moderate inhibitory effects on B. subtilis with MIC 25 µM compared to the positive control ampicillin with MIC 0.39 and 0.39 µM against MRSA and B. subtilis, respectively ( Table 2). The experiments show that the compounds could be used as spectral antifungal and antibacterial drug precursors for more in-depth research.

Fungal Material
The fungus strain Aspergillus flavus QQYZ in this work was isolated from a fresh blade of the mangrove plant Kandelia candel, collected from Huizhou in Guangdong province, China, and the strain was numbered as QQYZ. The fungal strain was identified by its sequence of the internal transcribed spacer (ITS) analysis of rDNA, and the following BLAST search result showed it was most similar (98%) to the strain Aspergillus flavus (compared to JQ776536.1). The sequence data of the fungus were submitted to GenBank, accession no. OK655763. The fungus is stored in our laboratory at Sun Yat-sen University with cryopreservation tubes at −20 • C.

Fermentation and Isolation
The fungus was cultured on rice solid medium in 60 Erlenmeyer flasks with a volume of 1 L each, containing 50 g rice and 50 g 0.3% brine, for 28 days after 5-day proliferation in 0.6 L potato dextrose broth. Then the culture medium and the mycothallus were soaked with MeOH and extracted with EtOAc after concentrations to 40 g of crude extract. The crude extract was separated by a silica gel column using petroleum ether and ethyl acetate with a ratio from 1:0 to 0:1 in 10 fractions (F1 to F10). Fraction F5 (1.93 g) was eluted on Sephadex LH-20 using CH 2 Cl 2 and MeOH (1:1 by volume) to yield three sub-fractions, and the second sub-fraction was separated by normal phase HPLC with isopropanol and n-hexane (35:65 by volume) to afford compound 1 (8.5 mg, t R 12.5 min) and compound 2 (12.1 mg, t R 8.0 min) ( Figure S19 Table 1

X-ray Crystallographic Analysis of Compounds 1 and 2
The single crystal of compounds 1 and 2 were acquired from a mixed solution of equal volumes of MeOH and CH 2 Cl 2 . The data were recorded by Agilent Xcalibur Nova singlecrystal diffractometer using Cu Kα radiation (λ = 1.5418 Å). The structures were solved by direct methods with the SHELXT software and refined by full-matrix least squares calculations. Non-hydrogen atoms were refined by anisotropic displacement parameters and hydrogen atoms were located on the calculated positions. The crystallographic data of 1 and 2 were deposited to the Cambridge Crystallographic Data Centre.

Antimicrobial Assays
The compounds were dissolved in DMSO and antifungal and antibacterial activities were assayed in 96-well plates by a serial dilution test in the range of 0.1-100 µM for the tested compounds, according to the methods in the previously published article [39,40]. Ketoconazole and ampicillin were used as positive controls for antifungal and antibacterial tests, and DMSO was used as a blank control, respectively.

Computation Methods
Initial conformational analysis was conducted by Spartan'14 software using the Merck Molecular Force Field (MMFF) method [41]. The conformation with the Boltzmann population greater than 1% were optimized at the B3LYP/6-31 + G (d,p) level with the density functional theory (DFT) and then performed to TDDFT at the B3LYP/6-311 + G (d,p) level to afford calculated ECD spectra in acetonitrile [42]. The calculated ECD spectra were generated using the SpecDis program (University of Würzburg) with sigma = 0.3 eV [43]. A 1D potential energy scan was performed on the dihedral angle C8-C7-C7 -C8 using the DFT method at the B3LYP/6-31g (d,p) level; the results were generated using GaussView 6 [44].

Conclusions
In this work, two asymmetric tetrahydroxanthone dimers with a 7,7 -linkage were described for the first time, reporting an unprecedented dimerization site of xanthones. The structures of the two new compounds were unambiguously identified by a range of methods, such as HRESIMS, NMR spectra, ECD calculations, and X-ray diffraction. Due to the rotation of the two units, part NMR signals in 2, missed at room temperature but obtained at a low temperature of 243 K, and 1D PES scans, were performed to verify the existence of axial chirality. Concurrently, it also represented an axial chirality with rapid rotation in non-biaryl compounds. The new compounds exhibited broad-spectrum and potential antifungal and antibacterial activities against C. gloeosporioides, F. oxysporum, F. oxysporum, C. musae, and C. albicans with MIC values in the range of 3.13-25 µM. Additionally, compound 1 possessed moderate antibacterial activities against MRSA and B. subtilis. This work increases the diversity of connection modes of xanthone dimers, which is valuable for the chemical diversity of xanthone dimers, it also provides more evidence to support mangrove endophytic fungi as a sustainable source of chemical diversity.
Author Contributions: Z.Z.: conceptualization, methodology, software, validation, investigation, chemical structure analysis, and writing-original draft preparation; W.Y.: methodology, software, resources, and chemical structure analysis; H.C.: methodology and software; R.C.: data curation and chemical structure analysis; C.L.: resources; G.Z.: software; B.W.: conceptualization, methodology, writing-review and editing, and funding acquisition, Z.S.: conceptualization, methodology, investigation, writing-review and editing, project administration and funding acquisition. All authors have read and agreed to the published version of the manuscript. Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed in this study are available within the manuscript and are available from the corresponding authors upon request.