New Polyphenols from a Deep Sea Spiromastix sp. Fungus, and Their Antibacterial Activities

Eleven new polyphenols namely spiromastols A–K (1–11) were isolated from the fermentation broth of a deep sea-derived fungus Spiromastix sp. MCCC 3A00308. Their structures were determined by extensive NMR data and mass spectroscopic analysis in association with chemical conversion. The structures are classified as diphenyl ethers, diphenyl esters and isocoumarin derivatives, while the n-propyl group in the analogues is rarely found in natural products. Compounds 1–3 exhibited potent inhibitory effects against a panel of bacterial strains, including Xanthomanes vesicatoria, Pseudomonas lachrymans, Agrobacterium tumefaciens, Ralstonia solanacearum, Bacillus thuringensis, Staphylococcus aureus and Bacillus subtilis, with minimal inhibitory concentration (MIC) values ranging from 0.25 to 4 µg/mL. The structure-activity relationships are discussed, while the polychlorinated analogues 1–3 are assumed to be a promising structural model for further development as antibacterial agents.


Introduction
The deep sea is a vast and relatively untapped source of unique molecular, structural and biological diversity with less than 2% of marine natural products reported in literature [1][2][3]. Life in the deep sea requires its inhabitants to adapt their biochemical machinery to cope with extreme conditions, involving exposure to high hydrostatic pressures, variable temperatures and low oxygen and light. The extremophilic organisms may have the potential to induce primary and secondary metabolic pathways to give rise to structurally unique metabolites. The recent advancements in marine technologies have allowed accessing the deep sea and to detect microbial activities [4,5], while screening of phylogenetically diverse and unique organisms from rare or extreme ecosystems in the deep ocean floor has been used to discover relevant bioactive metabolites. Deep sea derived natural products have emerged as a new frontier in drug discovery and development, leading to the identification of anti-tumor, anti-microtubule, anti-proliferative, photoprotective, antibiotic and anti-fouling compounds in the marine environment [6][7][8][9][10][11][12]. Despite the high scientific and commercial interest in the microbial ecology of these ecosystems, relatively little is known about the diversity of functional taxonomic groups of free-living microbes that occupy these niches as well as their biotechnological potential [13]. Fungi derived from deep water sediments have yielded an array of interesting new metabolites, including indole diketopiperazines, indole and quinazolinone alkaloids, sterigmatocystin derivatives, benzodiazepine alkaloids, polyketides, spiroditerpenoids, sesquiterpene quinones, sorbicillinoids, and trichoderones with strong bioactivities such as cytotoxic, antibiotic and antiviral effects.
In the course of our ongoing search for structurally unusual and bioactive secondary metabolites from deep sea derived microorganisms, a Spiromastix sp. fungus MCCC 3A00308 isolated from a sediment of South Atlantic at depth of 2869 m was examined chemically. Previously, a number of new depsidone-based spiromastixones A-O with potent antibacterial effects were isolated from the fermentation broth of this specimen [14]. Further examination of the minor components resulted in the isolation of 11 new diaryl derivatives, named spiromastols A-K (1-11) ( Figure 1). Herein, we report the isolation and structure elucidation of the new compounds and their antibacterial activities.

Results and Discussion
Spiromastol A (1) was isolated as a colorless oil ( Figure 1). Its molecular formula was deduced as C18H19Cl3O4 on the basis of the HRESIMS (m/z 403.0272 [M − H] − ) and NMR data, requiring eight degrees of unsaturation and containing three chlorine atoms. The IR absorption bands at 3408, 1651 and 1601 cm −1 suggested the presence of hydroxy and aromatic functionalities. The 1 H NMR spectrum displayed three exchangeable protons (δH 10.00, 9.99 and 9.73), two aromatic singlets at δH 5.97 (1H, s, H-1) and 6.57 (1H, s, H-3′), and the alkyl protons for four methylene and two methyl groups. The 13 C NMR spectrum exhibited a total of 18 carbon resonances, including 12 aromatic carbons for two phenyl moieties (rings A and B) and six alkyl carbons for two n-propyl groups, which were assigned by the COSY and HMBC correlations ( Figure 2). In regard to the substitution of the aromatic ring A, the HMBC interaction of H-1 with a n-propyl methylene (δC 35.6, C-7) allowed to assign the location of the n-propyl group vicinal to C-1 (δC 106.7). Additional HMBC interactions from H-1 to C-2 (δC 153.1), C-3 (δC 108.4), and C-5 (δC 114.8), and a weak correlation with C-4 (δC 150.3), H2-7 (δH 2.48, m) to C-1, C-5 and C-6 (δC 139.0), and a phenol proton at δH 10.00 (brs, OH-4) to C-3, C-4 and C-5 established a 3,5-disubstituted and 2,4-dioxygenated 6-propylphenyl segment, in which C-4 was hydroxylated. Similarly, the substitution of the aromatic ring B was established on the basis of the HMBC relationships. The observation of the HMBC interactions from the aromatic proton H-3′ to C-1′ (δC 132.7), C-2′ (δC 149.3), C-4′ (δC 151.5) and C-5′ (δC 110.4), the phenol proton at δH 9.73 (s, OH-2′) to C-1′, C-2′ and C-3′ (δC 102.7), and the other phenol proton at δH 9.99 (s, OH-4′) to C-3′, C-4′ and C-5′, in addition to the HMBC interactions of the second n-propyl protons, assigned a 5′-substituted and 1′-oxygenated 2′,4′-dihydroxy-6′-propylphenyl ring. Since two aromatic rings covered eight degrees of the molecular unsaturation, the connection of rings A and B was suggested through a C-C bond or an ether bond. The observation of NOE interaction between H-1 and OH-2′ ( Figure 2) assumed an ether linkage across C-2 and C-1′. Thus, the quaternary carbons C-3, C-5 and C-5′ were substituted by chlorine atoms. The molecular formula of spiromastol B (2) was determined as C18H18Cl4O4 by the HRESIMS (m/z 436.9873 [M − H] − ) and NMR data, indicating the presence of one more chlorine atom and the absence of a proton in comparison with those of 1. The NMR data of 2 were very similar to those of 1, with the exception of a quaternary carbon (δC 109.6, C-3′) of 2 to replace an aromatic methine of 1.
Spiromastol C (3) has a molecular formula of C19H20Cl4O4 as determined by the HRESIMS data (m/z 451.0042 [M − H] − ), containing a CH2 unit more than that of 2. The similar NMR data with the exception of the presence of methoxy resonances whose protons (δH 3.83, s) correlated to C-2′ (δC 150.5) in the HMBC spectrum, clarified compound 3 to be a 2′-methoxy analogue of 2. The downfield shifted C-1′ (Δ 3.8 ppm), C-2′ (Δ 4.1 ppm) and C-3′ (Δ 6.1 ppm) in comparison with the corresponding carbons of 2 further supported the methoxy substitution.
The molecular formula of spiromastol D (4) was determined as C19H22O6 based on the HRESIMS (m/z 345.1341 [M − H] − ) and NMR data. The 13 C NMR spectrum provided a total of 19 carbon resonances, including 12 aromatic carbons for two phenyl rings, a carboxylic carbon, and six alkyl carbons which were assigned to two n-propyl groups based on the COSY and HMBC data. The COSY spectrum displayed two aromatic spin systems of meta-couplings between δH 5.77 (1H, d, J = 2.3 Hz, H-3)/6.21(1H, d, J = 2.3 Hz, H-5) for ring A, and δH 6.27 (1H, d, J = 2.8 Hz, H-3′)/6.11 (1H, d, J = 2.8 Hz, H-5′) for ring B. In the aromatic ring A, the HMBC interactions from a phenol proton at δH 9.45 (s, OH-4) to C-3 (δC 98.7), C-4 (δC 158.8) and C-5 (δC 109.1) indicated C-4 to be hydroxylated. Additional HMBC interactions from H-5 to the methylene carbon at δC 35.5 and from H-3 and H-5 to C-1 (δC 115.7) and the carboxylic carbon at δC 169.5 through 4 JH-C coupling, revealed the position of a n-propyl group at C-6 (δC 141.9) and the carboxylic group at C-1. The oxygenated C-2 (δC 157.1) was assigned by the 2 JH-C coupling between H-3 and C-2 in the HMBC spectrum. The substitution of the second n-propyl group at C-6′ (δC 137.0) in the aromatic ring B was evident from the HMBC interaction between H-5′ and the methylene carbon at δC 32.0 (C-7′), while the NOE interactions between a phenol proton at δH 9.21 (s, OH-2′) and H-3′, and from the other phenol proton at δH 9.11 (s, OH-4′) to H-3′ and H-5′ assigned a 2′, 4′-dihydroxy-6′-propylphenyl ring. Since C-4 was positioned by a hydroxy group, the connection of the aromatic ring B at C-1′ (δC 132.6) with the aromatic ring A through an ether bond with C-2 or ester bond with C-10 was suggested. The observation of the carboxylic proton at δH 12.58 (brs) clarified C-10 to be an acidic group. Thus, the linkage of ring B with ring A though an ether bond across C-2 and C-1′ was assumed.
Spiromastol E (5) has a molecular formula of C20H24O6 as determined by the HRESIMS (m/z 359.1498 [M − H] − ) and NMR data, bearing a CH2 unit more than that of 4. Apart from 5 containing an additional methoxy group (δH 3.66, δC 56.0), the NMR data of both 5 and 4 were closely similar (Tables 1 and 2). The methoxy group of 5 was positioned at C-2′ (δC 153.2) on the basis of the HMBC relationship between the methoxy protons and C-2′. Thus, compound 5 was determined as a 2′-methoxylated analogue of 4. Table 1. The 1 H NMR data of spiromastols A-K (1-11) (δH ppm, J in Hz).   The molecular formula of spiromastol F (6) was determined as C19H21ClO6 by the HRESIMS (m/z 379.0951 [M − H] − ) and NMR data, with one chlorine atom more than that of 4. Comparison of the NMR data revealed that both compounds had the same partial structure of the aromatic ring B, whereas a quaternary carbon at δC 112.0 (C-5) in the aromatic ring A of 6 was recognized to replace the aromatic methine C-5 of 4. This finding reflected C-5 of 6 to be substituted by a chlorine atom. This assignment was supported by the HMBC correlations from H2-7 and OH-4′ (δH 10.10, s) to C-5, in association with the similar NMR data and HMBC relationships of the remaining resonances.

General Experimental Procedures
Optical rotations were measured on a Rudolph IV Autopol automatic polarimeter at 25 °C. UV spectra were measured on a Cary 300 spectrometer. IR spectra were measured on a Thermo Nicolet Nexus 470 FT-IR spectrometer. CD spectra were measured on a JASCO J-810 spectropolarimeter. 1 H, 13 C, and 2D NMR spectra were recorded on a Bruker Advance 400, 500, and 600 NMR spectrometers, respectively. Chemical shifts are expressed in δ (ppm) referenced to the solvent peaks at δH 2.50 and δC 39.5 for DMSO-d6, and δH 7.26 and δC 77.2 for CDCl3, respectively, and coupling constants are in Hz. HRESIMS spectra were obtained from Xevo G2 Q-TOF mass spectrometer. Materials for column chromatography (CC) involved silica gel Qingdao Marine Chemistry Co. Ltd.,Qingdao,China), ODS gel (50 μm, YMC, Japan) and Sephadex LH-20 (18-110 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden). Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) were used for TLC analysis. HPLC chromatography was performed on a Waters e2695 separation Module coupled with a Waters 2998 photodiode array detector and a semi-preparative reversed-phased column (YMC-packed C18, 5 μm, 250 mm × 10 mm) was used for purification.

Fungal Material and Fermentation
The fungal Spiromastix sp. MCCC 3A00308 was isolated from a deep ocean sediment, which was collected with TV-multicore in June 2011 from the South Atlantic Ocean at site S015-TVMC06 (GPS 13.75° W, 15.17° S) at a depth of 2869 m during the Comra 22nd oceanic cruise Leg 5. The fungus was identified as Spiromastix genus by ITS gene sequence analysis (GeneBank accession number KJ010057). The strain MCCC 3A00308 was deposited in the Marine Culture Collection Center (MCCC), Third Institute of Oceanography, State Oceanic Administration, Xiamen, China. The fungus Spiromastix sp. MCCC 3A00308 was cultured on PDA slants at 25 °C for 10 days. The fermentation was carried out in Erlenmeyer flasks (50 × 500 mL), each containing 100 g of rice, to which distilled H2O (140 mL) was added. The contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to about 30 °C, each flask was inoculated with 5 mL of the spore inoculum and incubated at 25 °C for 50 days.

Conclusions
In summary, this work described a group of new polyphenols with diverse scaffolds derived from deep sea derived fungus Spiromastix sp., while these findings provided additional evidence to support that the microorganisms from deep sea are a potential source for the discovery of chemical diversity. The potent antibacterial effects of 1-3 suggested that these compounds can be used for further lead modification.