Psathyrellanic Acid,a Monocyclic Diterpenoid From the Basidiomycete Psathyrella candolleana

Abstract

Psathyrelloic acid (1), a novel monocyclic diterpenoid, was isolated from the cultures of the basidiomycete Psathyrella candolleana. The structure was elucidated by means of spectroscopic methods. Psathyrelloic acid showed antibacterial activity against Staphylococcus aureus with an IC₅₀ value of 16 μg/mL.

Diterpenoids from fungi have structural and biological diversity, which attracted great attention of both organic and biological researchers. ^{1 -3} The basidiomycete Psathyrella candolleana, belonging to the family Psathyrellaceae, is commonly found on lawns or pastures in Europe and North America. It is also widely distributed in Yunnan and Sichuan provinces of China. It is used as a medicinal mushroom in Iraq, the extract of which could provide protection against DNA oxidative damage induced by doxorubicin. ^4,5 So far, the chemical constituents of this species are seldom reported. Only a number of biologically active guanacastane-type diterpenoids were isolated from the fermentation broths. ⁶ In our continuous search for structurally interesting and biologically active natural products from higher fungi, ^{7 -11} a novel diterpenoid, psathyrelloic acid (1, Figure 1), was isolated from the cultures of the fungus P. candolleana. The structure was identified by means of spectroscopic methods. Psathyrelloic acid (1) possessed a monocyclic system with 2 α,β-unsaturated ketone moieties which might be derived from the dolabellane-type skeleton. Its antibacterial activities against 4 strains were evaluated. Herein, the isolation, structural elucidation, and the biological activities of 1 are discussed.

Figure 1

Psathyrelloic acid (1) from Psathyrella candolleana.

Psathyrelloic acid (1) has a molecular formula C₂₀H₃₀O₅ as determined by positive high-resolution electrospray ionization mass spectrometry (HRESIMS), which showed a molecular ion peak at m/z 373.19843 (calcd for C₂₀H₃₀O₅Na, 373.19909), corresponding to 6 degrees of unsaturation. The infrared (IR) spectrum indicated the presence of hydroxy (3483 cm⁻¹), carbonyl (1708 cm⁻¹), and double bonds (1623, 1593 cm⁻¹). The 1D nuclear magnetic resonance (NMR) spectra (see Supplementary material), as well as the heteronuclear single-quantum coherence (HSQC) spectrum, revealed 4 methyls, 5 methylenes, 5 methines, and 6 quaternary carbons (Table 1). Three doubles (δ _C 119.2, 128.0, 132.7, 139.8, 140.5, 152.8) and 2 carbonyl carbons (δ _C 170.2, 170.9) suggested that compound 1 possessed 1 ring in the structure.

Table 1

¹H (600 MHz) and ¹³C (150 MHz) NMR Data of 1 ^a in Methanol-d ₄ (δ in ppm).

	δ H (J in Hz)	δ C, type
1		170.9, qC
2		128.0, qC
3	6.74 m	139.8, CH
4	2.20 m	41.1, CH2
5		45.5, qC
6a	1.55 m	37.0, CH2
6b	1.51 m
7a	1.65 m	24.4, CH2
7b	1.60 m
8	2.72 m	48.9, CH
9		152.8, qC
10	5.06 m	119.2, CH
11	3.06 dd (14.6, 9.2)	27.4, CH2
	3.01 dd (14.6, 4.1)
12		132.7, qC
13	6.75 m	140.5, CH
14	4.25 m	58.4, CH2
15	1.76 s	11.5, CH3
16	1.99 m	30.5, CH
17	0.88 d (6.8)	17.9, CH3
18	0.91 d (6.8)	20.7, CH3
19		170.2, qC
20	1.08 s	27.1, CH3

NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum coherence; HMBC, heteronuclear multiple bond correlation; COSY, correlation spectroscopy; ROESY, rotating frame nuclear Overhauser effect spectroscopy.

^aData were assigned by HSQC, HMBC, ¹H-¹H COSY and ROESY spectra.

On analyzing the ¹H-¹H correlation spectroscopy (COSY) spectrum, 4 fragments were established as shown in Figure 2. Based on the ¹H-¹H COSY data, a hydroxymethylene group for CH₂-14 was identified by the NMR (δ _H 4.25; δ _C 58.4) and mass spectroscopy (MS) data. Two downfield shifts for 2 olefinic protons at δ _H 6.74 and 6.75, as well as the corresponding carbon resonances at δ _C 139.8 (d, C-3) and 140.5 (d, C-13), together with 2 carbonyl carbons at δ _C 170.9 (s, C-1) and 170.2 (s, C-19), suggested the existence of 2 α,β-unsaturated carbonyl moieties. They were further identified as 2-methyl-2,3-unsaturated-1-carbonyl and 12,13-unsaturated-19-carbonyl, respectively, by the heteronuclear multiple bond correlation (HMBC) correlations from H-15 to C-1, C-2, and C-3, as well as from H-13 to C-19. According to the MS data, as well as the NMR data at δ _C 170.9 and 170.2, 2 carboxylic acid groups were identified for C-1 and C-19, respectively. In addition, a singlet for protons of a methyl at δ _H 1.08 (3H, s, H-20) showed key HMBC correlations to C-5, C-6, C-9, and C-4. This information, together with an HMBC correlation from H-8 to C-9, established a 5-membered carbon ring. This data, along with the long fragment as revealed by the ¹H-¹H COSY data, established an isopropyl connected to C-8. The third double bond between C-9 and C-10 determined the same. From HMBC correlations from H-11 to C-12 and C-19, the connection between C-11 and C-12 was established. Similarly, the connection between C-4 and C-5 was indicated by the HMBC correlations from H-4 to C-5 and from H-20 to C-4. So far, the planar structure of 1 was established as depicted.

Figure 2

Key 2D NMR correlations of 1. 2D NMR, two-dimensional nuclear magnetic resonance.

A ROESY experiment was used for the determination of relative configuration. At first, 3 double bonds were all elucidated as E form by the ROESY correlations of H-15/H-4, H-8/H-11, and H-11/H-14. In addition, a correlation of H-8 with H-7b, not observed for H-8 with H-7a, together with a correlation of H-20 with H-7a indicated that Me-20 and H-8 are in the opposite side. The structure of 1 was, finally, established as shown.

Structurally, compound 1 should be a diterpenoid related to a dolabellane-type framework. As shown in Scheme 1, a dolabellane-type diterpene backbone was built by cyclization of a geranylgeranyl pyrophosphate (GGPP). After oxidizations and cleavage, compound 1 was established. Our previous studies have demonstrated that the fungus P. candolleana could produce guanacastane-type diterpenoids which are featured with a 5/7/6 tricyclic framework. ⁶ Probably, compound 1 might be a by-product during the biosynthesis of guanacastane diterpenoids in this fungus.

Scheme 1

Proposed biosynthesis pathway for 1.

Compound 1 was evaluated for its antibacterial activities against Escherichia coli, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa by following the established bioassay method as reported previously. ¹² As a result, compound 1 showed weak antibacterial activity to S. aureus with an MIC of 16 µg/mL.

In summary, the chemical investigation on the cultures of the edible mushroom P. candolleana resulted in the isolation of a novel monocyclic diterpenoid, psathyrelloic acid (1). It showed significant antibacterial activity to S. aureus.

Experimental

General

Optical rotations were measured on a Rudolph Autopol IV polarimeter (Rudolph Technologies, Wilmington, USA). UV spectra were obtained on a UH5300 UV-VIS Double Beam Spectrophotometer (Hitachi High-Technologies (Shanghai) Co., Ltd., Shanghai, China). IR spectra were obtained by using a Shimadzu IRTracer-100 Fourier Transform Infrared spectrometer (Shimadzu, Kyoto, Japan) with KBr pellets. NMR spectra were acquired with a Bruker Avance III 600 instrument (Bruker, Germany). HSESIMS was measured on a Thermo Scientific Q Exactive Orbitrap MS system (Thermo Fisher, MA, USA). Silica gel (200-300 mesh and 80-100 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP-18 gel (40-75 µm, Fuji Silysia Chemical Ltd., Kasugai, Japan), and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) were used for column chromatography (CC). Fractions were monitored by thin-layer chromatography (Qingdao Marine Chemical Inc., China) and spots were visualized by 10% H₂SO₄ in methanol, in combination with Agilent 1260 series HPLC system (Zorbax SB-aq-C18 column, 5 µm, 6.4 × 150 mm). Preparative HPLC was performed on an Agilent 1260 series with a Zorbax SB-C18 (5 µm, 9.4 × 150 mm) column.

Fungal Material and Cultivation Conditions

Fruiting bodies of P. candolleana were collected at Jingdong, Yunnan Province, China, in 2003 and identified by Prof Zhu-Liang Yang (Kunming Institute of Botany). The voucher specimen (No. CGBWSHF00118) was deposited at the herbarium of Kunming Institute of Botany. ⁶ Culture medium was composed of glucose (5%), pork pepton (0.15%), yeast (0.5%), KH₂PO₄ (0.05%), and MgSO₄ (0.05%). Initial pH was adjusted to 6.0; the fermentation was first carried out on an Erlenmeyer flask for 6 days till the mycelium biomass reached to the maximum. Then it was transferred to rice medium at 24°C in dark culture for 40 days. Rice medium: 50 g of rice, 50 mL of water, placed in a 250 mL Erlenmeyer flask, sterilized at 121°C for 15 minutes, a total of 180 bottles.

Extraction and Isolation

The rice culture (9 kg) was extracted 4 times with EtOAc. The organic layer was evaporated to give a crude extract (90 g). Then it was subjected to silica gel CC (200-300 mesh) eluted with petroleum ether (PE)-Me₂CO gradient system to afford 8 fractions A to H. Fraction G (8 g) was first isolated by silica gel CC (200-300 mesh) eluted with PE-Me₂CO (2/1) to give subfractions G1 to G7. Fraction G5 (1.2 g) was separated by preparative MPLC with a reversed-phased C18 column (MeOH-H₂O, 10%-100%) to give fractions G5a to G5e; then fraction G5e (200 mg) was applied to Sephadex LH-20 CC (Me₂CO) and purified by HPLC (MeCN-H₂O, 20%-40%, 3 mL/min) to give 1 (2.1 mg).

Antibacterial Assay

The tested bacteria strains E. coli ATCC25922, S. aureus subsp. aureus ATCC29213, S. enterica subsp. enterica ATCC14028, and P. aeruginosa ATCC27853 were purchased from China General Microbiological Culture Collection Center (CGMCC). All these strains were cultured in Mueller Hinton broth (MHB) (Guangdong Huankai Microbial Sci. &Tech. Co., Ltd, Guangzhou, China) at 37°C overnight with shaking (200 rpm). A sample of each culture was then diluted 40-fold in fresh MHB broth and incubated with shaking (200 rpm) at 37°C for 2 to 3 hours. The resultant mid-log phase cultures were diluted to a concentration of 5 × 10⁵ CFU/mL, then 50 mL was added to each well of the compound-containing plates, giving a final compound concentration range of 128 or 50 mg/mL. Plates were covered and incubated at 37°C for 24 hours. Inhibition rates were determined using photometry at OD_{625 nm}. Penicillin G sodium salt (Biosharp, Shanghai, China) and ceftazidime (Shanghai Yuanye Biotechnologies, Shanghai, China) were used as positive inhibitor controls.

Psathyrelloic Acid (1)

Colorless oil, purity >95% (HPLC).

[α]²⁰ _D: +101.3 (c 0.5 MeOH).

IR (KBr) v _max: 3483, 2972, 2932, 1708, 1623, 1593, 1474, 1381, 1298, 1090, 1025, 960 cm⁻¹.

UV (MeOH) λ _max (log ε): 202 (3.61), 218 (3.70), 262 (3.16) nm.

¹H and ¹³C NMR data: see Table 1.

HRESIMS (pos.): m/z 373.19843 (calcd for C₂₀H₃₀O₅Na, 373.19855).