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

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.


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 OPEN ACCESS 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.  . 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. 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 H 3 -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). 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 H 3 -13 to C-7, C-11 and C-12, from H 3 -14 to C-4, C-5, C-6 and C-10, from H 3 -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/H 3 -15; H-12/H-11/H-7/H-6 and H-11/H 3 -13 established by 1 H, 1 H-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. 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 H 3 -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 H 3 -13 and H 3 -14 should both also be β-oriented deduced from the observation of NOE interactions between H-7 and H 3 -13, and H-6β and H 3 -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.

Results and Discussion
Comparison of the physicochemical properties and optical rotation data ([α] D 26 = +10° (c = 0.1, CHCl 3 )) with reported data allowed identifying the compound 2 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].

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, KH 2 PO 4 3 g, MgSO 4 1.5 g, citric acid 0.1 g, and thiamin hydrochloride 10 mg in 1.0 liter deionized H 2 O). 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.

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.

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.