New Prenylxanthones from the Deep-Sea Derived Fungus Emericella sp. SCSIO 05240

Four new prenylxanthones, emerixanthones A–D (1–4), together with six known analogues (5–10), were isolated from the culture of the deep-sea sediment derived fungus Emericella sp. SCSIO 05240, which was identified on the basis of morphology and ITS sequence analysis. The newstructures were determined by NMR (1H, 13C NMR, HSQC, HMBC, and 1H-1H COSY), MS, CD, and optical rotation analysis. The absolute configuration of prenylxanthone skeleton was also confirmed by the X-ray crystallographic analysis. Compounds 1 and 3 showed weak antibacterial activities, and 4 displayed mild antifungal activities against agricultural pathogens.


Introduction
Naturally occurringxanthones, dibenzo-γ-pyrone derivatives produced by higher plants, lichens, and fungi, contain different substituents on the two benzene rings, thus resulting in large structural OPEN ACCESS diversity [1,2]. These substituents are strongly dependent on their biosynthetic origins and modification reactions [3]. Prenylxanthones, an important subgroup of naturally occurring xanthones, exhibit diverse biological and pharmaceutical activities, because of their specific substitution patterns [4].In many conditions, activities are associated with prenylation of the xanthone skeleton [5].Various biological and pharmacological activities, such as antibacterial, antifungal, anti-inflammatory, antioxidant, and antitumor, have been reported for prenylxanthones, which make these compounds attractive for pharmaceutical applications [6].
Marine fungi are known to be a prolific source of biologically active natural products which might be useful for drug discovery [7]. As a special ecosystem, marine sediment provides an abundant of fungal resources, which yielded various secondary metabolites with novel structures and interesting biological activities [8]. The genus Emericella fungi could produce a remarkable diversity of secondary metabolites, such as indole alkaloids, prenylated polyketides, benzophenones, and xanthones, with interesting biological properties thus representing potential leads for the developing of new pharmaceutical agents [9,10]. As part of our research program to discover new natural products from marine deep-sea sediment fungi from South China Sea, we have isolated four new (1)(2)(3)(4) and six known (5)(6)(7)(8)(9)(10) prenylxanthones, from the culture of a deep-sea sediment derived fungus Emericella sp. SCSIO 05240.

Identification of the Fungus Strain
A fungal isolate from deep-sea sediment (3258 m) of the South China Sea displayed activity against bacteria and fungi in our previous screening tests. The cultural and morphological properties suggested that the isolate, termed SCSIO 05240, was a strain of Emericella (Figure 1a-d). Abundant conidiophores and cleistothecia occurred on PDA medium. Conidial heads were mostly short cylindrical. Vesicles were hemispherical and biseriate. Metulae were smoothing walled and cylindrical. Phialides were smooth walled and flask shaped, approximately equal in length to those of metulae. Conidia were olive green and globose to subglobose when mature, and spiny (Figure 1b,c). Cleistothecia were spherical after mature, and were typically surrounded by thickened large spherical envelop cells (Figure 1d). ITS1-5.8S-ITS2 sequence region (507 basepairs (bp), accession number KJ614489) of strain SCSIO 05240 was obtained and was found to be most similar to those of E.qinqixianii ML514, E. variecolor IFM 42010, and E. appendiculata IFM 54232, with sequence identity of 99%. A phylogenetic tree was constructed, using the neighbor-joining method based on similarity of a 506-bp consensus length of ITS1-5.8S-ITS2 sequence (Figure 2), and confirmed that strain SCSIO 05240 grouped most closely with E. variecolor IFM 42010. The ITS region sequence identity confirmed that strain SCSIO 05240 belonged to genus Emericella, and was designated as Emericella sp. SCSIO 05240.

Structure Elucidation
Four new prenylxanthones, emerixanthones A-D (1-4), together with six known analogues (5-10), were isolated and purified from the cultures of SCSIO 05240. Six known prenylxanthones were identified as shamixanthone (5) [11], tajixanthone hydrate (6) [11], ruguloxanthone A (7) [12], ruguloxanthone B (8) [12], tajixanthone (9) [13], and tajixanthone methonate (10) [11], by comparison with their 1 H and 13 C NMR with those reported (Figure 3). In order to confirm the absolute configuration of prenylxanthones skeleton, the X-ray crystallographic analysis of ruguloxanthone B (8) was carried out (Figure 4). It is the first X-ray crystallographic analysis of the prenylxanthones derivatives.    (Tables 1 and 2) and DEPT experiments suggested that the presence of 25 carbon signals, which were attributable to four methyls, three methylenes, six methines (including aromatic and chlorinated carbons), and nonprotonated (including one ketone carbonyl) carbons. The 1 H NMR spectroscopic data has showed resonances for three aromatic protons, with two (δ H 6.78, H-2 and δ H 7.55, H-3) exhibiting ortho coupling (J = 8.5 Hz) and the third (δ H 7.22, H-5) appearing as a singlet. The aromatic methyl substituent (δ H 2.35, s, H-24) was connected to position C-6, as supported by HMBC correlations of CH 3 -24 to C-5, C-6 and C-7. A chelated hydroxyl resonance at δ H 12.63 was assigned to the aromatic carbon atom C-1, as confirmed by HMBC correlations from OH-1 to C-1 and C-2. The OCHCHCH 2 O spin system, corresponding to the C-19/C-20/C-25 unit of 1, was consistent with a dihydropyran ring fused to an aromatic ring at the position of C-7 and C-8, which was also confirmed by the HMBC and 1 H-1 H COSY correlations showed in Figure 5. The two terminal olefinic proton signals, together with a methyl singlet at δ H 1.84 (3H, s, H-23), revealed that an isopropenyl moiety was connecting to the pyran ring at C-20 and was confirmed by HMBC correlation of H 3 -23 and H 2 -22 to C-20. These data indicated the same prenylxanthones skeleton as the known derivative ruguloxanthone B (8), whose absolute configuration has been confirmed by X-ray.   The isotopic ion peaks signals (3:1) in the ESIMS spectrum of 1 indicating the presence of a chlorine atom in the molecule. The isopentyl moiety connected to position C-4, which is different from ruguloxanthone B (8) and taxjixanthone hydrate (6), was deduced as 3-chloro-3-methyl-2-hydroxy butyl moiety, by the HMBC and 1 H-1 H COSY correlations showed in Figure 5. The chemical shifts of H-14a, H-14b/C-14 and H-15/C-15 (δ H /δ C 3.35, 2.76/31.9 and 3.84/78.5) also suggest C-15-OH, C-16-Cl replacement of 1, rather than C-15-Cl, C-16-OH moiety [9].
The configurations at C-20, C-25 of 1 are the same as those of ruguloxanthone B (8), whose absolute configuration has been confirmed by X-ray. The configuration at C-15 is same as taxjixanthone hydrate (6) , which were reported to as an R configuration at C-15 [12]. On the basis of this evidence as well as the comparison of CD spectrum of 1 with 2 ( Figure 6), 15S, 20S, 25R configuration of 2 was confirmed, as same as that of 1. The new prenylxanthone was named as emerixanthone B (2).
Compound 3 was obtained as yellow needle crystals and was assigned the molecular formula The NMR spectra revealed the same prenylxanthone skeleton of 3 as those of 1, 2 and 8. The 1 H and 13 C NMR spectra of 3 (Tables 1 and 2) were much similar to those of tajixanthone methonate (10) [11] except for the additional methoxy signal δ H 3.48 (3H, s, OCH 3 ) corresponding to C-15. One more methoxy signal δ H 3.29 (3H, s, OCH 3 ) was found to be connected to C-16, as same as that of 10. Thus, the side chain of 3 was deduced as 2, 3-dimethoxy-3-methyl butyl, which is confirmed by the HMBC spectrum. Compound 3 also exhibited negative specific optical rotation [−95.5 (c 0.0018, CHCl 3 )] in the same manner as 1. The comparison of CD spectrum of 3 with 1 and 2 ( Figure 6) also showed the same configuration of 3. This is the third new prenylxanthone,15,16-dimethoxytajixanthone hydrate, named as emerixanthone C (3).   (4). Tajixanthone hydrate (6) was stable and no change has been found subject to the extraction conditions, and taxjixanthone hydrate-15-formate was also reported as natural product [14]. Accordingly we believe that 4 is a true natural product, named as emerixanthone D (4).

Biological Activities of the Isolated Compounds
The isolated prenylxanthone derivatives 1-10 were evaluated for antibacterial, antifungal, and antitumor activities.

Discussion
Prenylxanthone derivatives have also been found in other Emericella fungi, such as E. rugulosa [12] and E. variecolor (Aspergillus variecolor) [11,13]. The biosynthesis of tajixanthone (9) and related metabolites of A. variecolor has been studied by incorporation experiments, and it provided further circumstantial evidence that this group of metabolites was biogenetically related to tajixanthone (9) and shamixanthone (5) [15]. The formation of shamixanthone (5) occurs by cyclodehydration of the dihydroxybenzophenone system present as a two hemiketal forms as arugosis A and B, could be an intermediate in the biosynthesis of tajixanthone, being formed through oxidative ring cleavage of a suitably substituted anthraquinone. Subsequent cyclodehydration and intramolecular 'ene' cyclization of the orthoprenyloxy aldehyde moiety would provide the xanthone nucleus and the substituted dihydropyran ring respectively. From this result, compounds 1-4 were obtained through the tajixanthone biosynthetic pathway under various modification of such as hydroxylation, oxidation, chlorination, acetylation, methylation, and rearrangement (Figure 7).

General
Optical rotations were measured with a perkine Elmer 341 polarimeter. Circular dichroism spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics, Ltd., Leatherhead, UK). NMR spectra were obtained on a Bruker AVANCE-500 spectrometer (Bruker, Karlsruhe, Germany) with TMS as internal standard, and chemical shifts were recorded as ä -values. HRESIMS were performed on a Q-Tof Micro mass spectrometer (Waters, Manchester, UK). Silica gel (100-200 and 200-300 mesh), and Sephadex LH-20 for column chromatography were purchased from Qingdao Marine Chemical Group Co. (Qingdao, China), and GE Healthcare (Uppsala, Sweden), respectively.All solvents used were of analytical grade (Tianjin Fuyu Chemical and Industry Factory, Tianjin, China).

Fungal Material and Fermentation
The strain SCSIO 05240 was isolated from a sediment sample (E 120°0.975′, N 19°0.664′) at the depth of 3258 m collected from an open voyage to the South China Sea in August 2007, and was deposited in the type culture collection of Center for Marine Microbiology, Research Network of Applied Microbiology, South China Sea Institute of Oceanology, CAS, Guangzhou, China. The fungus was identified by sequence analysis of the ITS1-5.8S-ITS2 sequence region and micromorphology as described previously [16].
The pure strain SCSIO 05240 colonies were sub-cultured in 100 mL Erlenmeyer flasks each containing 25 mL of malt media (20 g/L of malt extract, 1 g/L of peptone, and 30 g/L of sea salt). For a large scale culture, 25 mL of the seed culture was transferred to a 1000 mL Erlenmeyer flask containing solid rice medium (200 g rice, 200 mL of water, 3% sea salt), and incubated at 28°C under static stations and daylight. After one month, cultures from 30 flasks were harvested for isolation of compounds with antimicrobial activity.