A New Xanthone Glycoside from the Endolichenic Fungus Sporormiella irregularis

Abstract

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.

1. Introduction

Sporormiella is a genus belonging to the family of Sporormiaceae with more than 80 species distributed across boreal and temperate regions of the world [1,2,3,4]. Some species of the genus are saprobes, and others are endophytic in living plants, fungi, and lichens [5,6,7,8]. Previous chemical investigation of this genus led to isolation of polyketides (such as xanthones (sporormiellins A–C, sporormiellones A, B, microsphaeropsone A, brocaenol B) [5,9], chromones (corymbiferone C, corymbiferan lactone E, corymbiferone) [5], macrocyclic lactone (sporostatin) [10], organic acids (including zaragozic acid B, L731-127, L731-128, sporovexins A, B, and sporminarins A, B) [11,12,13]), triterpenoids (including FR171456, FR173945 [14,15], and (2aR,2a¹R,3S,5aR,6bS,9S,10aR,12bS)-9-hydroxy-2a¹,3,6b,10,10,12b-hexamethyl-1,2,2a,2a¹,3,5a,6,6b,7,8,9,10,10a,11,12,12b-hexadecahydro-4H-cyclopenta[de]naphtho[2,1-g]chromen-4-one [16]), steroids (including 22E-3,7-epoxy-5,10:8,9-disecoergosta-9(10),22-diene-5,8-dione, 22E-5α,6α-epoxyergosta-8(14),22-diene-3β,7α-diol, 22E-5α,6α-epoxyergosta-8(9),22-diene-3β,7α-diol, 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol, 22E-3β-hydroxy-5α,6α-epoxyergosta-22-en-7-one, 22E-ergosta-7,22-diene-3β,5α,6β-triol, 22E-6β-methoxyergosta-7,22-diene-3β,5α-diol, 22E-5α,8α-epidioxyergosta-6,22-dien-3β-ol and 22E-ergosta-4,6,8(14),22-tetraen-3-one [17]), and the nitrogenous compounds (including terezines A–D [18], similin B [19], sporovexin C [12], and (2Z,4E,6E)-3-hydroxy-N-(1,11a,11b-trihydroxy-3,6,9-trioxodecahydro-1H-pyrazino[1,2-a]pyrrolo[2,1-c]pyrazin-10-yl)octadeca-2,4,6-trienamide [20]).

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.

2. Results and Discussion

Compound 1 was obtained as a yellow amorphous powder. The quasi-molecular ion at m/z 451.1240 [M + H]⁺ by HRESIMS indicated the molecular formula of 1 was C₂₁H₂₂O₁₁ with 11 degrees of unsaturation. The ¹H-NMR spectrum (DMSO-d₆, Figure S1) displayed six exchangeable protons (δ_H 11.76 (1H, br s), 11.64 (1H, br s), 5.14 (1H, d, J = 4.9 Hz, 2′-OH), 5.01 (1H, d, J = 4.8 Hz, 3′-OH), 4.92 (1H, d, J = 5.1 Hz, 4′-OH), and 4.26 (1H, t, J = 5.6 Hz, 6′-OH)), three aromatic protons (δ_H 6.92 (1H, br s, H-3), 6.65 (1H, br s, H-15), and 6.58 (1H, s, H-7)), five sp³ methine protons (including one anomeric proton δ_H 4.84 (1H, d, J = 7.6 Hz, H-1′)), one sp³ methylene group (δ_H 3.60 (1H, ddd, J = 11.6, 5.6, 1.8 Hz, H-6′a) and 3.39 (1H, ddd, J = 11.6, 5.8, 5.6 Hz, H-6′b)), and two methyl groups (δ_H 3.92 (3H, s, 8-OCH₃), 2.39 (3H, br s, H-1)). The ¹³C-NMR and DEPT-135 spectra (Figure S2) of 1 showed 21 carbon signals including a ketone carbonyl (δ_C 183.6), twelve aromatic carbons (δ_C 160.1, 159.9, 157.6, 155.5, 149.3, 148.5, 125.1, 111.2, 107.9, 104.7, 101.3, and 95.5), two methyl carbons (including one oxygenated (δ_C 56.7)), and a set of hexose moiety carbons (δ_C 103.6, 77.3, 76.5, 74.2, 69.9, and 61.1). According to the analysis of coupling constants (³J_{H-2′,H-3′} = 8.6 Hz, ³J_{H-3′,H-4′} = 8.6 Hz, and ³J_{H-4′,H-5′} = 9.6 Hz), the analysis of ¹H-¹H COSY (Figure 1), and a comparison with the previously reported [21,22] ¹³C-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 δ_H 4.84 (1H, d, J = 7.6 Hz, H-1′). The connection position of Glu unit to aglycone was established at C-9 on the basis of the HMBC correlation from H-1′ to C-9. Combined with the molecular formula and chemical shifts of ¹H and ¹³C, 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-OCH₃ to C-8, and the key ROESY correlations between 8-OCH₃ and H-7/H-1′ (as shown in Figure 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 ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) data of compound 1 (in DMSO-d₆).

No.	1
	δC, Mult	δH, Mult, (J in Hz)	1H-1H COSY	HMBC	ROESY
1	21.9, CH3	2.39, br s	3, 15	2, 3, 15
2	149.3, qC
3	107.9, CH	6.92, br s	1, 15	1, 4, 12, 13, 15
4	155.5, qC
6	157.6, qC
7	95.5, CH	6.58, s		6, 8, 9, 11, 12	8-OCH3
8	160.1, qC
9	125.1, qC
10	148.5, qC
11	101.3 qC
12	183.6, qC
13	104.7, qC
14	159.9, qC
15	111.2, CH	6.65, br s	1, 3	1, 3, 13, 14
8-OCH3	56.7, CH3	3.92, s		8	7, 1′
6-OH *		11.76, br s
14-OH *		11.64, br s
1′	103.6, CH	4.84, d, (7.6)	2′	9	3′, 5′, 8-OCH3
2′	74.2, CH	3.32, overlapped	1′, 3′, 2′-OH	1′, 3′, 4′
3′	76.5, CH	3.24, td, (8.6, 4.8)	2′, 4′, 3′-OH	2′, 4′	1′, 5′
4′	69.9, CH	3.16, m	3′, 5′, 4′-OH	3′, 5′, 6′
5′	77.3, CH	3.09, ddd, (9.6, 5.8, 1.8)	4′, 6′a, 6′b	1′, 4′	1′, 3′
6′	61.1, CH2	3.60, ddd, (11.6, 5.6, 1.8), a 3.39, ddd, (11.6, 5.8, 5.6), b	5′, 6′b, 6′-OH 5′, 6′a, 6′-OH
2′-OH		5.14, d, (4.9)	2′
3′-OH		5.01, d, (4.8)	3′
4′-OH		4.92, d, (5.1)	4′
6′-OH		4.26, t, (5.6)	6′a, 6′b

*: The assignment maybe exchanged.

.

Compound 2 was obtained as a yellowish amorphous powder. The quasi-molecular ion at m/z 323.0527 [M + Na]⁺ by HRESIMS indicated the molecular formula of 2 was C₁₆H₁₂O₆ with 11 degrees of unsaturation. The ¹H-NMR spectrum (DMSO-d₆, Figure S7) displayed two exchangeable protons (δ_H 13.23 (1H, br s), 12.33 (1H, brs)), four aromatic protons (δ_H 7.20 (1H, d, J = 2.4 Hz, H-9), 6.99 (1H, d, J = 2.4 Hz, H-7), 6.88 (1H, br s, H-3), and 6.67 (1H, br s, H-15)), and two sp³ methyl groups (δ_H 3.96 (3H, s, 8-OCH₃), 2.41 (3H, s, H-1)). The ¹³C-NMR and DEPT-135 spectra (Figure S8) showed 16 signals, assigned two carbonyl carbons (a ketone one (δ_C 179.2) and a carboxylic acid/ester one (δ_C 168.8)), twelve sp² aromatic carbons, and two methyl carbons (including an oxygenated one (δ_C 56.6)). Combined with the molecular formula and chemical shifts of ¹H and ¹³C, the key HMBC correlations from H-1 to C-2/C-3/C-15, from H-3 to C-4/C-13/C-15, from H-15 to C-3/C-13/C-14, from H-7 to C-5/C-8/C-9/C-11, from H-9 to C-7/C-8/C-10/C-11, from 8-OCH₃ to C-8, and the key ROESY correlations between 8-OCH₃ and H-7/H-9 (as shown in Figure 1) established the planar structure of 2, which was a methyl esterified derivative of calyxanthone [24] at C-8. Therefore, the structure of 2 was elucidated as the same as that of the known compound (calyxanthone-8-methyl ether). The assignments of NMR data of 2 are provided in

Table 2. The ¹H-NMR (400 MHz) and ¹³C-NMR (100 MHz) data of 2 (in DMSO-d₆).

No.	2
	δC, Mult	δH, Mult, (J in Hz)	1H-1H COSY	HMBC	ROESY
1	21.9, CH3	2.41, br s	3, 15	2, 3, 15
2	148.8, qC
3	107.2, CH	6.88, br s	1, 15	1, 4, 13, 15
4	155.2, qC
5	168.8, qC
6	136.5, qC
7	111.8, CH	6.99, d, (2.4)	9	5, 8, 9, 11	8-OCH3
8	164.8, qC
9	101.2, CH	7.20, d, (2.4)	7	7, 8, 10, 11	8-OCH3
10	157.6, qC
11	109.7, qC
12	179.2, qC
13	105.9, qC
14	160.5, qC
15	111.2, CH	6.67, br s	1, 3	1, 3, 13, 14
8-OCH3	56.6, CH3	3.96, s		8	7, 9
5-OH *		13.23, br s
14-OH *		12.33, br s

*: The assignment maybe exchanged.

.

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 ¹H-NMR and ¹³C-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]. The resulting emodin will be enzymatically transformed into intermediate a by oxidative ring opening between C-4 and C-5 [26,29], and subsequent dehydration will give intermediate b. Compound 2 may be derived from intermediate b via methylation. After decarboxylation and oxidation, intermediate b may transform into intermediate c, and 1 may be derived from intermediate c via methylation and glycosylation [26].

3. Materials and Methods

3.1. Chemicals

l-cysteine methyl ester hydrochloride, o-tolyl isothiocyanate, d-glucose (d-Glc), l-glucose (l-Glc), and DMSO-d₆ were purchased from Sigma-Aldrich Chemical Co. Ltd. (Saint Louis, MO, USA). Methanol (MeOH) was purchased from Yuwang Industrial Co. Ltd. (Yucheng, China). Formic acid (HCOOH) was obtained from Kemiou Chemical Reagent Co. Ltd. (Tianjin, China).

3.2. 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-d₆: 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 C₁₈ column (4.6 mm × 250 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 C₁₈ column (10.0 mm × 250 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).

3.3. 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 H₂O (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.

3.4. 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–H₂O (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–H₂O 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–H₂O to yield 19 subfractions (W2a to W2s). Subfraction W2m (50.0 mg) was purified by semi-preparative HPLC with MeOH–H₂O–HCOOH (80:20:0.1, v/v) at a flow rate of 3 mL/min to yield 2 (4.0 mg). Subfraction W2i (66.1 mg) was purified with semi-preparative HPLC using MeOH–H₂O–HCOOH (60:40:0.1, v/v) at flow rate of 3 mL/min to yield 3 (9.0 mg).

Sporormielloside (1) (Figure 2): Yellow amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\text{D}}^{25}$ = –46.3 (c = 0.08, CHCl₃–MeOH = 1:2); UV (MeOH) λ_max (log ε) 206 (4.66), 232 (4.24), 256 (4.42), 336 (4.13) nm; IR (KBr) υ_max 3421, 2906, 2360, 2339, 1632, 1608, 1515, 1370, 1267, 1209, 1160, 1122, 1071, 1024, 552 cm⁻¹; ESIMS (positive): m/z 473 [M + Na]⁺, 923 [2M + Na]⁺; HRESIMS (positive): m/z 451.1240 [M + H]⁺ (calcd. for C₂₁H₂₃O₁₁, 451.1240); ¹H- and ¹³C-NMR data see Table 1.

Calyxanthone-8-methyl ether (2) (Figure 2): Yellowish amorphous powder; UV (MeOH) λ_max (log ε) 202 (4.53), 236 (4.62), 251 (4.51), 302 (4.37) 351 (3.96) nm; IR (KBr) υ_max 3446, 3248, 2360, 2335, 1739, 1650, 1600, 1571, 1504, 1455, 1423, 1394, 1273, 1217, 1179, 1154, 1128, 1016, 829, 687 cm⁻¹; ESIMS (positive): m/z 301 [M + H]⁺, 323 [M + Na]⁺; ESIMS (negative) m/z 299 [M − H]⁻, 255 [M ‒ COOH]^‒; HRESIMS (positive): m/z 323.0527 [M + Na]⁺ (calcd. for C₁₆H₁₂O₆Na, 323.0532); ¹H- and ¹³C-NMR data see Table 2.

Endocrocin (3) (Figure 2): Yellow amorphous powder; UV (MeOH) λ_max (log ε) 203 (4.55), 226 (4.65), 375 (3.50), 440 (4.26) nm; IR (KBr) υ_max 3377, 2361, 2339, 1715, 1671, 1622, 1384, 1255, 1206, 1171, 756 cm⁻¹; ESIMS (negative) m/z 313 [M − H]⁻; HRESIMS (negative): m/z 313.0345 [M − H]⁻ (calcd. for C₁₆H₉O₇, 313.0348).

3.5. Acid Hydrolysis

Acid hydrolysis was performed according to the method described by Tanaka et al. with standard monosaccharides [23]. Compound 1 (1.0 mg) was hydrolyzed with 2 M of HCl for 1 h at 90 °C. After it was extracted with EtOAc twice, the H₂O layer was evaporated in vacuo to furnish a monosaccharide residue using a rotary evaporator. The residue was dissolved in pyridine (1.0 mL) containing L-cysteine methyl ester hydrochloride (1.0 mg) and heated at 60 °C. After 1 h, 10 μL of o-tolyl isothiocyanate was added to the reaction mixture and further reacted at 60 °C for 1 h. Then, the reaction mixture was directly analyzed by the Dionex HPLC system and detected by an UV detector (at 254 nm). Analytical HPLC was performed on the Phenomenex Gemini C₁₈ column with isocratic elution of CH₃CN–H₂O (40:60, v/v) for 40 min at a flow rate of 0.8 mL/min. The standard monosaccharides of D-Glc and L-Glc were subjected to the same method.

4. Conclusions

Xanthones are commonly found in higher plants, fungi, and lichens [30,31]. However, only eight xanthone aglycones have been isolated from Sporormiella. Xanthone glycosides are mainly isolated from plants such as Gentianaceae and Guttiferae [32,33] but are rarely reported from fungi, except for Phomopsis sp. ZH76 [34], Paecilomyces cinnamomeus BCC 9616 [35], and Aschersonia coffeae Henn. Bcc 28712 [36]. The isolation of sporormielloside (1), which is the first report for xanthone glycoside isolated from Sporormiella. Owing to a lack of any references to calyxanthone-8-methyl ether (2) in the literature, moreover, the assignments of NMR data of 2 are provided here for the first time.