Dihydroberkleasmin A: A New Eremophilane Sesquiterpenoid from the Fermentation Broth of the Plant Endophytic Fungus Pestalotiopsis photiniae

Abstract

Dihydroberkleasmin A (1), a new ester-substituted sesquiterpenoid related to the eremophilane class, together with the known compound berkleasmin C (2), were isolated from the fermentation broth of the plant endophytic fungus Pestalotiopsis photiniae. The structure of dihydroberkleasmin A (1) was elucidated by extensive spectroscopic analysis. The stereochemistry was assigned by comparison of the NMR spectroscopic data with those of berkleasmin A.

1. Introduction

Fungi of the genus Pestalotiopsis (Amphisphaeriaceae), as one class of the most widely distributed endophytic fungi, are common in their distribution, and many are saprobes, while others are either pathogenic or endophytic to living plants [1,2,3,4]. Since discovery of the anticancer agent taxol from an endophytic fungal strain of the genus Pestalotiopsis [5,6], interest in searching for bioactive compounds from this fungal genus has increased considerably. Up to date, about 300 species of the genus Pestalotiopsis have been recorded in China, but only about 10% of these species referred to chemical investigations. Previous chemical studies of some species of this genus have afforded a variety of bioactive metabolites [7,8,9,10,11,12,13,14,15,16,17,18]. In the course of our research on bioactive metabolites of the genus Pestalotiopsis in China, the present study was undertaken to investigate the chemical constituents of the culture broth of Pestalotiopsis photiniae isolated from the branch of Podocarpus macrophyllus in Hainan (People’s Republic of China), and have led to the isolation of a new eremophilane sesquiterpenoid named dihydroberkleasmin A (1) and one known compound, berkleasmin C (2). Details of the isolation and structural elucidation of 1 are reported herein.

Figure 1. The structures of compounds 1, 2 and berkleasmin A.

Figure 1. The structures of compounds 1, 2 and berkleasmin A.

2. Results and Discussion

Compound 1 was obtained as an optically active white powder, [α]_D^22.0 = +70° (c = 0.1, MeOH) that gave a quasi-molecular ion peak at [M+Na]⁺ m/z 545.3458 in the HR-ESI-MS (positive mode), consistent with a molecular formula of C₃₀H₅₀O₇ (calcd. for C₃₀H₅₀O₇Na, 545.3454), requiring six degrees of unsaturation. The IR spectrum revealed absorption bands of double bond (1,604 cm⁻¹), hydroxyl (3,424 cm⁻¹) and carbonyl (1,736 cm⁻¹) groups. There were 30 signals observed in the ¹³C-NMR spectrum (Table 1). Analysis of the ¹³C-NMR, DEPT, and HSQC spectra revealed that 1 contained one carbonyl carbon, seven oxygenated carbons, two olefinic carbons, eight methylene carbons, five methine carbons, one quaternary carbon, and six methyl carbons. Analysis of the ¹H-NMR spectrum (Table 1) indicated the presence of six methyl signals including one tertiary methyl [δ_H 1.62 (s), 1.15 (s), 1.13 (d, J = 7.2 Hz), 1.05 (d, J = 6.6 Hz), 0.89 (d, J = 6.7 Hz), 0.87 (t, J = 7.2 Hz)], one olefinic proton signal [δ_H 5.23 (d, J = 10.1 Hz)], two oxygenated methylene protons signals [δ_H 3.95, 3.54 (m), 3.63, 3.34 (m)] and three oxygenated methine proton signals [δ_H 4.50 (m), 4.09 (d, J = 7.4 Hz ), 3.27 (s)]. By careful analysis of NMR data, we found that the spectral data of 1 were similar to those of berkleasmin A recently reported from the saprobic fungus Berkleasmium nigroapicale [19], and this suggested that 1 has a tricyclic sesquiterpene core attached to a long-chain acid through an ester linkage. The distinct differences between 1 and berkleasmin A are: the chemical shifts value at C-11 and C-13 of 1 [δ_C 42.7 (d, C-11), 16.0 (q, C-13)] are absent in berkleasmin A [δ_C 151.2 (s, C-11), 104.3 (t, C-13)]. In addition, the chemical shifts value at C-7 (δ_C 48.3) and C-12 (δ_C 72.3) in 1 were shifted downfield compared to berkleasmin A [δ_C 44.6 (s, C-7), 69.9 (t, C-12)] because of replacement of the exomethylene group in berkleasmin A by a methyl group in compound 1.

Table 1. ¹H-(600 MHz) and ¹³C-NMR (150 MHz) data for 1 in CDCl₃, and the literature data for berkleasmin A [19].

No.	δH	δC	No.	δH	δC
1	4.50 (m)	74.8 (d)	1'		175.3 (s)
2	1.86, 1.77 (m)	28.5 (t)	2'	2.71 (dq, 7.3, 7.4)	42.6 (d)
3	1.44, 1.76 (m)	25.7(t)	3'	4.09 (d, 7.4)	79.1 (d)
4	1.58 (m)	38.9 (d)	4'		137.5 (s)
5		36.2 (s)	5'	5.23 (d, 10.1)	130.8 (d)
6	α 1.30 (t, 13.0)β 1.64 (dd, 13.0, 6.8 )	37.2 (t)	6'	2.56 (m)	40.8 (d)
7	1.70 (m)	48.3 (d)	7'	1.09, 1.29 (m)	31.3 (t)
8		102.3 (s)	8'	1.21–1.28 (m)	27.2 (t)
9	3.27 (s)	62.4 (d)	9'	1.21–1.28 (m)	29.4 (t)
10		62.8 (s)	10'	1.21–1.28 (m)	31.8 (t)
11	1.80 (m)	42.7 (d)	11'	1.21–1.28 (m)	22.6 (t)
12	3.54, 3.95 (m)	72.3 (t)	12'	0.87 (t, 7.2)	14.1 (q)
13	1.05 (d, 6.6)	16.0 (q)	13'	1.13 (d, 7.2)	15.0 (q)
14	1.15 (s)	15.3 (q)	14'	1.62 (s)	12.2 (q)
15	0.89 (d,6.7)	15.1 (q)	15'	3.34, 3.63 (m)	66.5 (t)

Further interpretation of the HMBC spectrum showed the following long-range correlations (Figure 2): from H-2' to C-1', C-3', C-4' and C-13', from H-3' to C-1', C-2', C-4', C-5', C-13' and C-14', from H-5' to C-3', C-14' and C-15', from H-6' to C-4', C-5', C-7' and C-15', from H₃-13' to C-1', C-2' and C-3', from H-14' to C-3', C-4' and C-5', from H-15' to C-5' and C-7'.

Table 1. ¹H-(600 MHz) and ¹³C-NMR (150 MHz) data for 1 in CDCl₃, and the literature data for berkleasmin A [19].

No.	δH	δC	No.	δH	δC
1	4.50 (m)	74.8 (d)	1'		175.3 (s)
2	1.86, 1.77 (m)	28.5 (t)	2'	2.71 (dq, 7.3, 7.4)	42.6 (d)
3	1.44, 1.76 (m)	25.7(t)	3'	4.09 (d, 7.4)	79.1 (d)
4	1.58 (m)	38.9 (d)	4'		137.5 (s)
5		36.2 (s)	5'	5.23 (d, 10.1)	130.8 (d)
6	α 1.30 (t, 13.0)β 1.64 (dd, 13.0, 6.8 )	37.2 (t)	6'	2.56 (m)	40.8 (d)
7	1.70 (m)	48.3 (d)	7'	1.09, 1.29 (m)	31.3 (t)
8		102.3 (s)	8'	1.21–1.28 (m)	27.2 (t)
9	3.27 (s)	62.4 (d)	9'	1.21–1.28 (m)	29.4 (t)
10		62.8 (s)	10'	1.21–1.28 (m)	31.8 (t)
11	1.80 (m)	42.7 (d)	11'	1.21–1.28 (m)	22.6 (t)
12	3.54, 3.95 (m)	72.3 (t)	12'	0.87 (t, 7.2)	14.1 (q)
13	1.05 (d, 6.6)	16.0 (q)	13'	1.13 (d, 7.2)	15.0 (q)
14	1.15 (s)	15.3 (q)	14'	1.62 (s)	12.2 (q)
15	0.89 (d,6.7)	15.1 (q)	15'	3.34, 3.63 (m)	66.5 (t)

Further interpretation of the HMBC spectrum showed the following long-range correlations (Figure 2): from H-2' to C-1', C-3', C-4' and C-13', from H-3' to C-1', C-2', C-4', C-5', C-13' and C-14', from H-5' to C-3', C-14' and C-15', from H-6' to C-4', C-5', C-7' and C-15', from H₃-13' to C-1', C-2' and C-3', from H-14' to C-3', C-4' and C-5', from H-15' to C-5' and C-7'.

Figure 2. The fragments and selected HMBC correlations of 1.

Figure 2. The fragments and selected HMBC correlations of 1.

The above spectral evidence, along with the proton spin system: H-3'/H-2' and H-2'/H₃-13'; H-5'/H-6'/H-7'/H-8'/H-9'/H-10'/H-11'/H₃-12' and H-6'/H-15' deduced from ¹H, ¹H-COSY (Figure 3) correlations, led to the establishment of the partial structure 1a (Figure 2). In addition, HMBC spectrum also showed the long-range couplings from H-1 to C-1', C-3 and C-10, from H-7 to C-6, C-8, C-9 and C-11, from H-9 to C-1, C-7, C-8 and C-10, from H-11 to C-6, C-7 and C-12, from H-12 to C-7, C-8, C-11 and C-13, from H₃-13 to C-7, C-11 and C-12, from H₃-14 to C-4, C-5, C-6 and C-10, from H₃-15 to C-3, C-4 and C-5. These spectral data, coupling with the following correlations: H-1/H-2/H-3/H-4/H₃-15; H-12/H-11/H-7/H-6 and H-11/H₃-13 established by ¹H,¹H-COSY correlations (Figure 3), gave rise to another partial structure 1b (Figure 2). The ester bond linkage, C-1'-O-C-1, between fragments 1a and 1b was clearly determined by the HMBC correlation of H-1 with C-1', which permitted the construction of the planar structure of 1 as shown in Figure 2.

Figure 3. The ¹H, ¹H-COSY and key selected NOESY correlations of 1.

Figure 3. The ¹H, ¹H-COSY and key selected NOESY correlations of 1.

The relative configuration of 1 was elucidated by analysis of the partial NOESY data and comparison chemical shifts with berkleasmins A-E and cryptosphaerolide [19,20]. The same relative stereochemistry of C-1, C-4, C-5, C-8, C-9, C-10, C-2', C-3' and C-4' in 1 as in berkleasmins A-E were deduced from the very similar carbon and proton chemical shifts. The β-oriented configuration of H-7 and H₃-15 was indicated by the observation of NOE interactions (Figure 3) between H-6β (δ_H 1.64 (dd, 13.0, 6.8)) and H-7, and H-6α and H-4, respectively. The relative configuration of H₃-13 and H₃-14 should both also be β-oriented deduced from the observation of NOE interactions between H-7 and H₃-13, and H-6β and H₃-14, respectively. The E-configuration of trisubstituted olefin was assigned by NOESY correlations from H-3' to H-5', and from H-6' to H-14'. Because of some significant signal overlap, we tried to crystallize of 1 in different solvents but finally failed to obtain crystals. Due to small quantity sample, we can not further determine the relative configuration of 1 by chemical methods. Finally, the relative configuration of remaining chiral centers of 1 except for C-6' were determined by comparison chemical shifts with berkleasmins A-E. Unfortunately, the relative configuration of C-6' remains unsigned through only spectroscopic analysis. Through comparison the NMR data of 1 with that of berkleasmin A, the absolute configurations of C-1, C-8, C-2', and C-3' in 1 as berkleasmin A were determined to be 1R, 8S, 2'R, 3'S.

Comparison of the physicochemical properties and optical rotation data ([α]_D²⁶ = +10° (c = 0.1, CHCl₃)) with reported data allowed identifying the compound2 as berkleasmin C [19], recently reported from the saprobic fungus Berkleasmium nigroapicale and shown to possess cytotoxicity against anti-cancer cell-lines (NCI-H187, MCF-7, and KB) and antimalarial activities. The relative and absolute configurations of 2 were from literature [19].

3. Experimental

3.1. General

Optical rotations: Perkin-Elmer 341 spectropolarimeter. IR spectra: Perkin-Elmer 577 spectrometer; KBr pellets; in cm^-1. NMR spectra: Bruker AM-600 spectrometer; δ in ppm, J in Hz; Me₄Si as internal standard, measured in CDCl₃. FT-MS spectra: Bruker Apex-Ultra 7.0 T spectrometer, in m/z. Column chromatography (CC): silica gel (200~300 mesh, Yantai Zhi Fu chemical Co., Ltd., People’s Republic of China), RP-18 (12 nm, S-50 um, YMC Co., Ltd., Japan), TLC: silica gel GF₂₅₄ plates (Yantai Zhi Fu chemical Co., Ltd, People’s Republic of China) and Sephadex LH-20 gel (25~100 μm, GE Healthcare Co., Ltd., Sweden).

3.2. Fungal Material and Cultivation Conditions

Pestalotiopsis photiniae was isolated from the branches of Podocarpus macrophyllus in Hainan, People’s Republic of China, in April, 2008, and identified by Professor Jing-Ze Zhang, Institute of Biotechnology, Zhejiang University. The isolate was assigned the accession number L328 in the culture collection at College of Life Science, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University. The fungal strain was cultured on slants of potato dextrose agar (CPDA ) at 28 °C for 7 days, and then inoculated into a 500 mL Erlenmeyer flask containing 100 mL of medium (glucose 20 g, potato (peeled) 200 g, KH₂PO₄ 3 g, MgSO₄ 1.5 g, citric acid 0.1 g, and thiamin hydrochloride 10 mg in 1.0 liter deionized H₂O). The final pH of the media was adjusted to 6.5 before sterilization. After 7 days of incubation at 28 °C on rotary shakers at 150 rpm, 25 mL of culture liquid were transferred as seed into each 1,000 mL Erlenmeyer flask containing 250 mL of medium and static fermentation was carried out on a rotary shaker for 30 days.

3.3. Extraction and Isolation

The culture broth (20 L) was extracted three times with ethyl acetate. Evaporation of the solvent in vacuo gave a brown oily residue (18.0 g), which was subjected to column chromatography (silica gel), eluted with petroleum ether/acetone [100:0, 98:2, 95:5, 90:10, 80:20, 50:50 (v/v)] to afford six fractions Fr. 1-6. Fr. 5 (3.0 g) eluted with petroleum ether/acetone (80:20) was further purified by CC (silica gel; CHCl₃/acetone, 8:1) to afford eight fractions Fr. 5.1-5.8. Fr. 5.3 (500 mg) was subjected to Sephadex LH-20 chromatography (CHCl₃/MeOH, 1:1) to afford compounds 1 (3.0 mg) and 2 (2.5 mg).

Dihydroberkleasmin A (1): Isolated as white powder, [α]_D²²= +70° (c = 0.1, MeOH). IR (KBr) v_max: 3,424 (OH), 1,736 (C=O), 1,604 (C=C) cm⁻¹. ¹³C- (150 MHz, CDCl₃) and ¹H-NMR (600 MHz, CDCl₃): see Table 1. Positive ion ESI-MS m/z (%): 545 [M+Na]⁺ (21), 1,068 [2M+Na+H]⁺(7). Positive ion HR-ESI-MS [M+Na]⁺ m/z 545.3458 (calcd for C₃₀H₅₀O₇Na, 545.3454).

4. Conclusions

In summary, we have isolated a new eremophilane-type sesquiterpene, named dihydroberkleasmin A (1), together with one known compound, berkleasmin C (2), from the culture broth of Pestalotiopsis photiniae. Eremophilane-type sesquiterpenes, including those with similar skeletons such as berkleasmins A-C, exist widely as constituents of various plants, while there have been several reports as fungal secondary metabolites mostly from family Xylariaceae. There has been no reported about eremophilane-type sesquiterpenes from the genus Pestalotiopsis.