Unusual dimeric tetrahydroxanthone derivatives from Aspergillus lentulus and the determination of their axial chiralities

The research on secondary metabolites of Aspergillus lentulus afforded eight unusual heterodimeric tetrahydroxanthone derivatives, lentulins A−H (2−9), along with the known compound neosartorin (1). Compounds 1−6 exhibited potent antimicrobial activities especially against methicillin-resistant Staphylococci. Their absolute configurations, particularly the axial chiralities, were unambiguously demonstrated by a combination of electronic circular dichroism (ECD), Rh2(OCOCF3)4-induced ECD experiments, modified Mosher methods, and chemical conversions. Interestingly, compounds 1–4 were the first samples of atropisomers within the dimeric tetrahydroxanthone class. Further investigation of the relationships between their axial chiralities and ECD Cotton effects led to the proposal of a specific CD Exciton Chirality rule to determine the axial chiralities in dimeric tetrahydroxanthones and their derivatives.


Result and Discussion
Structural elucidation. The crude extract of Aspergillus lentulus, obtained from its rice cultures, was purified through silica gel and ODS column chromatography (CC), as well as preparative HPLC to yield compounds 1-9 (Fig. 1).
Lentulin A (2) was obtained as a yellow powder. Its molecular formula, C 34 O 15 Na). UV absorption at 337 nm suggested the presence of a long conjugated moiety and IR absorptions at 3451 and 1740 cm −1 indicated the existence of hydroxy and carbonyl groups. The typically doubled NMR data (Supporting Information Tables S1 and S2), especially the presence of two conjugated ketone carbonyls (δ C 187.8, 187.2) and two enolic groups (δ H 14.11, 1 H, s and δ C 178.4, 101.1; δ H 14.02, 1 H, s and δ C 179.3, 100.2), revealed the dimeric tetrahydroxanthone skeleton of 2. Further comparison of its 1D NMR data and HMBC correlations ( Fig. 2) with those of neosartorin (1) 16 denoted the identical planar structure between them (Fig. 1), possessing 2,4′ -linked blennolide C (unit A) 17 and 5-acetyl blennolide A (unit B) 18 .
Lentulin C (4) had the same molecular formula C 34 H 32 O 15 (HRESIMS ion at m/z 703.1637) as 3, and was deduced to be its isomer from their similar 1D NMR data (Supporting Information Tables S1 and S2). The aS* configuration was also demonstrated by the NOE correlations of OH-1/OCH 3 -12′ and CH 3 -3/H-3′ .
HRESIMS (m/z 721.1736 [M + Na] + ) gave the molecular formula C 34 H 34 O 16 for lentulin F (7), with one more H 2 O unit than 5. An intensive comparison of its NMR data (Supporting Information Tables S1 and S2) with those of 5 confirmed the γ-hydroxy butyric acid moiety in 7 17 , notably through one degree loss of unsaturation and the lack of HMBC correlation from H-5 to C-8. The relative configuration of 7 was confirmed same as 5 from their identical NOE correlations.
8 and 9 were the derivatives of 5 and 6 with the cleavages of γ-lactones and the formations of γ-hydroxy butyric acid methyl esters, which were denoted by the decreases of unsaturation and HMBC correlations from the additional methoxyls to C-8. Similar to 5 and 6, the relative configurations of COOCH 3 -10 in 8 and 9 were confirmed as βand α-orientation respectively. Their axial configurations were also assigned same as those of 5 and 6.
The absolute configuration (aR, 5S, 10R, 5′ S, 6′ S, 10′ R) of neosartorin was determined by ECD calculation, which also predicted the approximate mirror image CEs of its atropisomer 16 . In the ECD spectrum of 2 ( Fig. 4), the CEs well matched the calculation for neosartorin atropisomer with aS, 5S, 10R, 5′ S, 6′ S, 10′ R configuration, proving that their CEs were governed by axial chiralities. The similar mirror images CEs were also found for 3 and 4 (Fig. 4). The identical CEs between 3 and 1 indicated the aR configuration in 3, while 4 was assigned as the atropisomer of 3. A Rh 2 (OCOCF 3 ) 4 -induced ECD experiment (Supporting Information Figure S1) suggested the 5S configuration in 4 according to the Bulkiness rule 21 , confirming its absolute configuration as aS, 5S, 10S, 5′ S, 6′ S, 10′ R. Interestingly, although 3 was the epimer of 1 with different central chiralities at C-10, the signs of their CEs were still the same, also suggesting the domination of axial chiralities in ECD spectra. So were compounds 4 and 2.
To avoid any ambiguity and demonstrate the absolute configurations of other derivatives, series of chemical conversions from 1 into 8, 3 into 9, and 7 into 5 were performed (Figs 5 and 6) 17,22 . Succeeding derivations of 8 and 9 by (R)-and (S)-methoxyphenylacetic acid (MPA) allowed the determination of their absolute configurations 22 . The negative Δ δ H(R-S) values of OCH 3 -13 and H-7 as well as the positive Δ δ H(R-S) values of OCH 3 -12 and H-9 ( Fig. 7) confirmed 5S absolute configurations in both 8 and 9. In combination with above chemical conversions, the 5S configurations in the other compounds were also revealed.
A plausible biosynthetic pathway was shown in Fig. 1. The oxidization and reduction of chrysophanol from the two paths gave units A and B 6,23 . Unit C (epi-blennolide C) was afforded via ring open and closure of unit A and both of them could convert into γ-lactone products through the retro-Dieckmann cyclization, which further yielded other ring cleavage intermediates 5,6,17 . Interestingly, all the biosynthetic modifications were occured on monomeric unit A, while the derivatization of unit B was limited to 5-acetylation that could prevent the further retro-Dieckmann cyclization. Meanwhile, the dimers were regularly formed by unit B with diverse path A units. These patterned dimerisations together with the specific monomeric modifications, indicated the enzymatic nature of these reactions, rather than the radical coupling 24,25 . CD Exciton Chirality method. Tetrahydroxanthone monomers possessed two distinct kinds of chromophores ( Fig. 8), namely 1-arylpropenone (330 nm) and benzoyl (230 nm). Their corresponding ECD CEs at    above wavelengths were not split 18 . But for the axially linked dimers, their obviously split CEs suggested the two chromophores interacted with each other, and the opposite but not mirror image ECD spectrums ( Fig. 4) revealed axial chiralities governed chromophore spatial positions to affect ECD CEs 26,27 .
When watching parallel to the chiral axes, the two chromophores' rotary manners were identical to those of the CD Exciton Chirality rule. In particular, the anticlockwise manner of two 1-arylpropenone chromophores 16 led to negative exciton couplets centered at around 330 nm like 1 and 3 (Fig. 8a), otherwise positive like 2 and 4 ( Fig. 8b). This deduction was simultaneously proved by the split CEs at 230 nm caused by two benzoyl chromophores in the same rotary manners. Interestingly, due to the cleavages of one 1-arylpropenone chromophore in 5-9, their exciton couplets at 330 nm disappeared (Fig. 9), also suggesting the domination of chromophore interactions in ECD CEs. For further verification, different dimeric tetrahydroxanthone analogues were checked, which accurately revealed the same result as determined by X-ray analysis, chemical conversions or ECD calculations (Supporting Information Table S3) 15,17,28,29 .
The CD Exciton Chirality method has been applied to some axially chiral determinations before, such as those of binaphthyl, biarylic dihydronaphthopyranone 30,31 , and bis(naphtho-γ-pyrone) 32 . However, a very few exceptions were reported due to their complicated chromophores and non-negligible intense magnetic transition dipole moments 33,34 .
Theoretically, the rotational strengths R α and R β of α and β excited states of one exciton couplet can be defined as Equations (1) and (2) 33,35 (μ, electric transition dipole moments; m, magnetic transition dipole moments): The first terms (μμ terms) were the rotational strengths caused by electric transition dipole moments μ and the second terms (μm terms) described the rotational strengths obtained by the combined electric and origin-independent magnetic transition moments. For the common π − π * excitation chromophores such as p-substituted benzoates, naphthoates, and anthroates, their internal magnetic transition moments (m iao , m jao ) were small and negligible, so were the μm terms 35,36 . The remaining terms (μμ terms) were the  Antimicrobial Assays. Using broth microdilution method 37 , compounds 1-9 were evaluated for their antimicrobial activities against a panel of pathogenic microbes, including multidrug resistant clinical strains. As collated in Table 1, compounds 1-6 showed moderate to significant antibacterial activities against four strains of Gram-positive and three strains of Gram-negative bacteria. 3 was the most potent antibiotic among these compounds, especially against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE). In view of the weakly active 5 and 6, as well as the inactive ring-opening derivatives (7-9), 8-OH might be important for their antibacterial activities.

Conclusion.
In summary, our study on fungal metabolites afforded nine rare 2,4′ -linked heterodimeric tetrahydroxanthone derivatives with attractive antibacterial activities. Compounds 1-4 were first isolated as two pairs of atropisomers, which along with other derivatives, provided great samples to reveal the dominant role of axial chiralities in CEs. The specific CD Exciton Chirality method was therefore proposed to determine the axial chiralities in dimeric tetrahydroxanthones and their derivatives.

Methods
General experimental procedures. Optical rotations were measured on a Jasco P-1020 polarimeter (Jasco, Tokyo, Japan). A UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan) was applied for the measurement of UV spectra. ECD and IR data were collected on Jasco J-810 spectrometer and Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany), respectively. Preparative HPLC was performed on a Shimadzu LC-8A system equipped with Shim-pack RP-C 18 column (10 μm, 200 mm × 20 mm i.d., Shimadzu, Tokyo, Japan), using a binary channel UV detector at the wavelengths of 210 and 330 nm, respectively. With TMS as internal standard, NMR data were recorded on a Bruker AVIII-500 NMR instrument ( 1 H NMR, 500 MHz; 13    Fungal material. The tubers of Pinellia ternata (Araceae) were collected from the suburb of Nanjing, Jiangsu, People's Republic of China in May, 2014. After surface sterilization with 75% ethanol and 1% NaClO, the tubers were cut into small pieces, which were put on the potato dextrose agar to afford the title strain. From morphological and microscopic characteristics, this fungus was identified as Aspergillus lentulus, which was further reinforced by its internal transcribed spacer (ITS) and 18S rDNA sequences with 100% identity to the reported one (GenBank accession No. HE578064.1). The fungus was cultivated on potato dextrose agar (PDA) at 28 °C for 7 days. Then 16 pieces of the agar were transformed into four 250 mL Erlenmeyer flasks (containing 80 mL potato dextrose liquid medium), which were incubated at 28 °C, and 120 rpm for 6 days to prepare seed culture. A total of 15 Erlenmeyer flasks (500 mL), each containing 80 g of rice and 120 mL of tap water, were used for solid fermentation. After being sterilized at 115 °C for 30 minutes, the flasks were incubated with 20 mL of seed cultures to cultivate at 28 °C for 30 days.
Extraction and isolation. The    Absolute configuration of 5-secondary alcohol in 4. To a CH 2 Cl 2 solution of 4 (0.5 mg/mL), 0.8 mg of Rh 2 (OCOCF 3 ) 4 was added and the first induced ECD spectrum was recorded immediately. The following spectra were measured every 5 minutes until reaching a stationary state. The absolute configuration of 5-sencondary alcohol was determined by the induced CE at around 350 nm.