A New Xanthone Glycoside from the Endolichenic Fungus Sporormiella irregularis

A new xanthone glycoside, sporormielloside (1), was isolated from an EtOAc extract of an endolichenic fungal strain Sporormiella irregularis (No. 71-11-4-1), along with two known xanthones (2, 3). Their structures were determined by detailed spectroscopic analysis (IR, MS, and 1D- and 2D-NMR), a chemical method, and a comparison of NMR data with closely related compounds previously reported. According to the structures of isolated compounds, their plausible biosynthetic pathway was deduced.

In our previous chemical investigation of this genus Sporormiella, a series of xanthones (sporormiellins A-C, sporormiellones A, B, and microsphaeropsone A) were isolated from S. minima (No. 66-3-4-2) [9].Notably, sporormiellin A is the first discovered tetrahydrofuran-fused furochromone with an unprecedented tetracyclic skeleton.As a part of our continuing search for more xanthones, a chemical investigation of another species (S. irregularis (No. 71-11-4-1)) from this genus was carried out, which led to isolation of a new xanthone glycoside, sporormielloside (1), along with two known biogenetically related compounds, calyxanthone-8-methyl ether (2) and endocrocin (3).Compound 2 is a known compound; however, there is no reference reporting its NMR data.Therefore, the details of the isolation and structural elucidation of these isolated compounds are reported herein.
Molecules 2016, 21, 764 2 of 8 In our previous chemical investigation of this genus Sporormiella, a series of xanthones (sporormiellins A-C, sporormiellones A, B, and microsphaeropsone A) were isolated from S. minima (No. 66-3-4-2) [9].Notably, sporormiellin A is the first discovered tetrahydrofuran-fused furochromone with an unprecedented tetracyclic skeleton.As a part of our continuing search for more xanthones, a chemical investigation of another species (S. irregularis (No. 71-11-4-1)) from this genus was carried out, which led to isolation of a new xanthone glycoside, sporormielloside (1), along with two known biogenetically related compounds, calyxanthone-8-methyl ether (2) and endocrocin (3).Compound 2 is a known compound; however, there is no reference reporting its NMR data.Therefore, the details of the isolation and structural elucidation of these isolated compounds are reported herein.1), and a comparison with the previously reported [21,22] 13 C-NMR data of the glucopyranoside unit, the hexose moiety of 1 was identified as glucopyranoside (Glu).After acid hydrolysis and derivatization of 1, the HPLC analysis revealed the presence of D-glucoses and compared them with derivatives obtained by the same method with standard monosaccharides [23].The Glu unit in 1 was attached to the aglycone via a β-linkage on the basis of the coupling constant of the anomeric proton located at  1).Therefore, the structure of 1 was elucidated as shown in Figure 2, which was a new xanthone glycoside called sporormielloside.The assignments of all proton and carbon resonances are provided in Table 1.*: The assignment maybe exchanged.
Based on the structural features of compounds 1-3, the plausible biosynthetic pathway of them was deduced (Scheme 1).The C 16 -octaketide produced by non-reducing polyketide synthase undergoes cyclization to yield atrochryone carboxylic acid [26], which is either autoxidized to endocrocin (3) or undergoes dehydration first followed by decarboxylation and spontaneous oxidation to give emodin [27,28]  Compound 3 was obtained as a yellow amorphous powder.The quasi-molecular ion at m/z 313.0345 [M − H] − by HRESIMS indicated the molecular formula of 3 was C16H10O7 with 12 degrees of unsaturation.The 1 H-NMR and 13 C-NMR spectrum (DMSO-d6) are the same as that of endocrocin [25].
Based on the structural features of compounds 1-3, the plausible biosynthetic pathway of them was deduced (Scheme 1).The C16-octaketide produced by non-reducing polyketide synthase undergoes cyclization to yield atrochryone carboxylic acid [26], which is either autoxidized to endocrocin (3) or undergoes dehydration first followed by decarboxylation and spontaneous oxidation to give emodin [27,28]

General Experimental Procedures
UV data were recorded using a JASCO V-550 UV/vis spectrometer (Jasco International Co. Ltd., Tokyo, Japan).IR data were recorded on a JASCO FT/IR-480 plus spectrometer (Jasco International Co. Ltd.).Optical rotations were measured on a JASCO P1020 digital polarimeter (Jasco International Co. Ltd.).The ESIMS spectra were performed on a Bruker amaZon SL mass spectrometer (Bruker Daltonics Int., Boston, MA, USA), and the HRESIMS spectra were obtained on a Waters Synapt G2 mass spectrometer (Waters Corporation, Milford, MA, USA).1D and 2D NMR spectra were acquired with Bruker AV 400 spectrometers (Bruker BioSpin Group, Faellanden, Switzerland) using the solvent signals (DMSO-d 6 : δ H 2.50/δ C 39.5) as internal standards.The analytical HPLC was performed on a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 DAD, an Ultimate 3000 Column Compartment, an Ultimate 3000 autosampler (Thermo Fisher Scientific Inc., Sunnyvale, CA, USA), and an Alltech (Grace) 2000ES evaporative light scattering detector (Alltech International Inc., Vienna, VA, USA) using a Phenomenex Gemini C 18 column (4.6 mm ˆ250 mm, 5 µm) (Phenomenex Inc., Los Angeles, CA, USA).Semi-preparative HPLC was performed on a Dionex HPLC system, which was equipped with an Ultimate 3000 pump, and an Ultimate 3000 RS variable wavelength detector using a Phenomenex Gemini C 18 column (10.0 mm ˆ250 mm, 5 µm) (Phenomenex Inc.).The medium pressure liquid chromatography (MPLC) was performed on ODS (60-80 µm, YMC Co. Ltd., Tokyo, Japan) and equipped with a dual pump gradient system, a UV preparative detector, and a Dr Flash II fraction collector system (Lisui E-Tech Co. Ltd., Shanghai, China).The organic solvent was evaporated with an EYELA rotary evaporator N-1100 system (Tokyo Rikakikai Co. Ltd., Shanghai, China).

Fungus Material
The strain numbered as 71-11-4-1 was isolated from the lichen Usnea mutabilis Stirt, which was collected in Zixishan Mountain, Yunnan province, China, in November 2006.The strain was identified as S. irregularis based on the morphological characters by one of our authors (L.-D.G.).The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25 ˝C for 5 days.Agar plugs were used to inoculate nine Erlenmeyer flasks (250 mL), each containing 100 mL of potato dextrose broth (PDB).The nine flasks of the inoculated media were incubated at 25 ˝C on a rotary shaker at 200 rpm for five days to prepare the seed culture.Fermentation was carried out in 30 Erlenmeyer flasks (500 mL), each containing 70 g of rice.Distilled H 2 O (105 mL) was added to each flask, and the rice was soaked overnight before autoclaving at 121 ˝C for 30 min.After cooling to room temperature, each flask was inoculated with 10 mL of the spore inoculum and incubated at 25 ˝C for 40 days.

Extraction and Isolation
The culture was extracted four times with EtOAc, and the organic solvent was evaporated in a vacuum to afford the dry crude extract (29.05 g).The crude extract was dissolved in 90% v/v aqueous MeOH (500 mL) and partitioned against the same volume of cyclohexane to afford a cyclohexane fraction (C, 13.42 g) and an aqueous MeOH fraction (W, 12.38 g).The aqueous MeOH fraction (W, 12.38 g) was separated by MPLC eluting with MeOH-H 2 O (30:70, 50:50, 70:30, and 100:0, v/v) to afford four fractions (W1 to W4).Fraction W3 (753.4 mg) was further separated by MPLC with a gradient of MeOH-H 2 O to yield 9 subfractions (W3a-W3i) and 1 (11.7 mg).Fraction W2 (1.14 g) was further separated by MPLC with a gradient of MeOH-H 2 O to yield 19 subfractions (W2a to H 4.84 (1H, d, J = 7.6 Hz, H-1 1 ).The connection position of Glu unit to aglycone was established at C-9 on the basis of the HMBC correlation from H-1 1 to C-9.Combined with the molecular formula and chemical shifts of 1 H and 13 C, the structure of aglycone was established based on the key HMBC correlations from H-1 to C-2/C-3/C-15, from H-3 to C-4/C-12/C-13/C-15, from H-15 to C-3/C-13/C-14, from H-7 to C-6/C-8/C-9/C-11/C-12, from 8-OCH 3 to C-8, and the key ROESY correlations between 8-OCH 3 and H-7/H-1 1 (as shown in Figure