Saccharomonopyrones A–C, New α-Pyrones from a Marine Sediment-Derived Bacterium Saccharomonospora sp. CNQ-490

Intensive study of the organic extract of the marine-derived bacterium Saccharomonospora sp. CNQ-490 has yielded three new α-pyrones, saccharomonopyrones A–C (1–3). The chemical structures of these compounds were assigned from the interpretation of 1D, 2D NMR and mass spectrometry data. Saccharomonopyrone A (1) is the first α-pyrone microbial natural product bearing the ethyl-butyl ether chain in the molecule, while saccharomonopyrones B and C possess unusual 3-methyl and a 6-alkyl side-chain within a 3,4,5,6-tetrasubstituted α-pyrone moiety. Saccharomonopyrone A exhibited weak antioxidant activity using a cation radical scavenging activity assay with an IC50 value of 140 μM.


Introduction
Actinobacteria are known as an abundant source of novel secondary metabolites comprising over 45% of all bioactive microbial metabolites known [1]. The recent discovery of numerous taxonomically unique marine actinomycetes, along with the isolation of structurally unprecedented secondary metabolites from these strains, illustrates marine actinomycetes as a promising source for the discovery of new natural products [2]. Saccharomonospora, a genus in the actinomycete family Pseudonocardiaceae, was first described in 1971 [3,4]. Members of the genus Saccharomonospora are interesting because they originate from diverse habitats and play an important role in the primary degradation of plant material by attacking hemicellulose [5]. Previous chemical investigations of members of this genus have led to the isolation of bioactive secondary metabolites, such as antibiotic AB 65, sakyomicin E, saccharonol A, antimicrobial saccharonol B, and piericidin A 3 [6,7]. As part of our ongoing research for new secondary 2 of 8 metabolites from marine actinobacteria, a Saccharomonospora bacterial strain CNQ-490 was documented. In a previous study, this strain was found to produce a novel cytotoxic alkaloid, lodopyridone A [8]. Further chemical investigation of this strain has now yielded three new natural products of the α-pyrone class. Herein, we report the isolation and structure elucidation of saccharomonopyrones A-C (1-3) along with their biological activities ( Figure 1).
Mar. Drugs 2017, 15, 239 2 of 8 our ongoing research for new secondary metabolites from marine actinobacteria, a Saccharomonospora bacterial strain CNQ-490 was documented. In a previous study, this strain was found to produce a novel cytotoxic alkaloid, lodopyridone A [8]. Further chemical investigation of this strain has now yielded three new natural products of the α-pyrone class. Herein, we report the isolation and structure elucidation of saccharomonopyrones A-C (1-3) along with their biological activities ( Figure 1).

Results and Discussion
Saccharomonopyrone A (1) was obtained as a white amorphous powder. An high-resolution electrospray ionization mass spectrometry (HR-ESIMS) peak [M + H] + at m/z 241.1446 (calcd for 241.1434) indicated the molecular formula C13H20O4, which implied four degrees of unsaturation. UV absorption at 288 nm and IR absorptions at 3402, 1672, and 1569 cm −1 indicated the presence of an α-pyrone moiety.

Results and Discussion
Saccharomonopyrone A (1) was obtained as a white amorphous powder. An high-resolution electrospray ionization mass spectrometry (HR-ESIMS) peak [M + H] + at m/z 241.1446 (calcd for 241.1434) indicated the molecular formula C 13 H 20 O 4 , which implied four degrees of unsaturation. UV absorption at 288 nm and IR absorptions at 3402, 1672, and 1569 cm −1 indicated the presence of an α-pyrone moiety.
Saccharomonopyrone B (2) was isolated as an amorphous white powder. Its molecular formula C 14  Pyrones are a well-known class of microbial secondary metabolites and are found to have a wide range of biological activities such as anti-inflammatory [9], anticancer [10], antimicrobial [11,12], anti-obesity [13,14], and antibiotic activities [15]. Previous studies on microbial sources have shown that the main producer of α-pyrones is fungi, but α-pyrones are also produced by plants, animals, and bacteria. Marine bacteria have also yielded α-pyrones with interesting bioactivities, examples of which are antibiotic Sch419560 [15], germicidins, and cytotoxic violapyrones. A recent study also expanded our knowledge for the first time that pyrones can be used as signaling molecules of the bacterial cell-cell communications in the soil bacterium Pseudomonas [16]. Bacteria monitor other bacteria in their living environment by producing and responding to signaling molecules. This led to a strategy to prevent pathogenicity by blocking bacterial communication in their environment [17][18][19].
Saccharomonopyrones A-C (1-3) were tested on various assays such as monoamine oxidase (MAO) inhibitory, acetylcholinesterase (AChE) inhibitory, β-site amyloid precursor protein cleaving enzyme 1 (BACE1), anti-osteoporosis, cytotoxicity, anti-tyrosinase, and antibacterial activities. However, the compounds did not display any significant biological activities in these assays. Interestingly, saccharomonopyrone A (1) showed weak antioxidant activity measuring free radical scavenging activity and cation radical scavenging activity in assays with IC 50 values of 911 µM and 140 µM, respectively.
Saccharomonopyrone A (1) has an unusual ethylbutyl ether moiety attached at C-6. There are no reports of an ether moiety attached at C-6 within the α-pyrone class. Some similar molecules with ethyl-methyl sulfide and propyl-methyl sulfide groups were obtained as bioengineered products [20]. A similar compound possessing an ethyl chain with an acetyl end group was obtained during the synthesis of β-polyketones [21]. In addition, another naturally occurring and structurally similar compound is known to possess a propyl chain with a methyl carbonyl end attached at the C-6 position [22]. However, the butoxyethyl side chain in 1 is unprecedented within this class of natural products.
Nocapyrone R and violapyrone B are the most structurally related compounds of saccharomonopyrone B (2) [11,23]. The only difference is that nocapyrone R has a methoxy group at C-3 and violapyrone B has no methyl group on C-5. Violapyrone I, structurally the most similar natural product of saccharomonopyrone C (3), also lacks the methyl group on C-5 [10].
The genus Saccharomonospora (Family Pseudonocardiaceae), with eleven known species to date, has been known since 1971 [24]. Saccharomonospora sp. CNQ-490 is a unique sediment-derived strain which produces the structurally unprecedented lodopyridones A-C [8,25]. Furthermore, by genome mining, this strain has been shown to possess a full biosynthetic pathway to produce a new antibiotic taromycin A through direct cloning and refactoring methods [26]. Saccharomonopyrones A-C, and previously reported natural products, lodopyridones and taromycin A, illustrate the versatile secondary metabolites producing ability of this strain. The application of a recent genetic modification method for the biosynthetic gene cluster of this strain to introduce a methyl group in natural products could lead to the successful production of new secondary metabolites [27].

General Experimental Procedures
The UV spectra were recorded in methanol (MeOH) on a UVS-2100 (Scinco, Seoul, Korea). The IR spectra were obtained using a Varian Scimitar Series. The NMR spectra were obtained using a Varian Inova NMR spectrometer (500 and 125 MHz for 1 H and 13 C NMR, respectively, Varian Inc., Palo Alto, CA, USA), using the signals of the residual solvent as internal references and δ H 2.50 and δ C 39.5 ppm for dimethyl sulfoxide-d 6 (DMSO). High Resolution Mass spectra were determined by a JMS-AX505WA mass spectrometer (Jeol Ltd., Tokyo, Japan). Low-resolution LC-MS data were analyzed using an Agilent Technologies 6120 quadrupole LC/MS system with a reversed-phase column (Phenomenex Luna C 18 (2) 100 Å, 50 mm × 4.6 mm, 5 µm, Phenomenex, Torrance, CA, USA) at a flow rate of 1.0 mL/min (Agilent Technologies, Santa Clara, CA, USA). Column chromatographic separation was performed using C 18 (40-63 µm, ZEOprep 90, Zeochem, Zurich, Switzerland) with a gradient solvent of MeOH and water (H 2 O). The fractions were purified using reversed-phase HPLC (Waters 600S controller with 996 PDA detector (Waters Corporation, Milford, MA, USA), Phenomenex Luna C 18 (250 mm × 10 mm, 5 µm) column (Phenomenex, Torrance, CA, USA)) with a mixture of acetonitrile (ACN) and H 2 O at flow rate of 2.0 mL/min. All chemicals were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and used without further purification.

Strain Isolation and Fermentation
Actinomycete strain Saccharomonospora sp. CNQ-490 was collected from a deep sea sediment sample 2 km west of the Scripps pier, in La Jolla, CA, USA. The 16S rDNA sequence of this strain showed modest identity with the genus Saccharomonospora (accession number: KF301601). Strain CNQ-490 was cultured in 40-L scale using 2.5-L Ultra Yield (Thomson Instrument Company, Oceanside, CA, USA) flasks, each containing 1 L of the medium (10 g/L of soluble starch, 2 g/L of yeast, 4 g/L of peptone, 10 g/L of CaCO 3 , 20 g/L of KBr, 8 g/L of Fe 2 (SO 4 ) 3 ·4H 2 O dissolved in 1000 mL artificial seawater) at 25 • C with shaking at 150 rpm. After 7 days, the broth was extracted with ethyl acetate (added ratio 1:1 of volume). The ethyl acetate layer was separated and dried with anhydrous sodium sulfate. The organic solvent was removed to yield 2.5 g of the organic extract.

Extraction and Isolation
The crude organic extract was loaded on the C 18 resin and fractionated by reversed-phase C 18

Bioactivity Assays
MAO inhibitory assay [28], AChE inhibitory assay [29], BACE1 [25], anti-osteoporosis assay [30], and anti-tyrosinase assay [31] were performed following the previously published methods. Cytotoxicity tests were performed on two human kidney cancer cell lines, A498 and ACHN renal cancers, according to previously published methods [32]. Antibacterial assays were performed against seven bacterial strains including four Gram-positive (Staphylococcus epidermidis ATCC 12228, Kocuria rhizophila ATCC 9341, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 65381) and three Gram-negative (Escherichia coli ATCC 11775, Salmonella typhimurium ATCC 14028, Klebsiella pneumoniae ATCC 4352) strains following a previously published method [25]. The antioxidant activity was performed using the 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) as described previously with slight modification [33]. The DPPH solution (0.45 mM) was prepared daily in a 20-mL conical tube and kept in the dark at 4 • C. The DPPH solution (120 µL) was added to 60 µL of sample, control, or standard solution in 70% ethanol at different concentrations. The solutions were mixed, covered, and allowed to react in the dark for 15 min; afterward, the absorbance at 517 nm was read. Ascorbic acid was used as a positive control (IC 50 21.02 ± 0.82 µM). Data are presented as the mean values ± standard deviation (SD) of three measurements. The free radical scavenging activity of each solution was then calculated as the percent inhibition according to the following equation: Scavenging rate (%) = [A (blank) − A (sample)]/A (blank) × 100 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) cation radical scavenging activity of the compounds were tested using the spectroscopic method described by Roberta et al. [34]. The ABTS cation radical (ABTS+) was acquired by reacting 7 mM solution of ABTS with 2.45 mM potassium persulfate reaction. The mixture was left to stand in the dark at room temperature for 12-16 h before use. Prior to the assay, the ABTS radical cation solution was diluted with ethyl alcohol to an absorbance of 0.750 ± 0.05 at 734 nm. The ABTS+ solution was then added to each sample, standard, and control solution. Ascorbic acid was used as a positive control (IC 50 13.01 ± 0.21 µM). Data are presented as the mean values ± standard deviation (SD) of the three measurements. The extent of decolorization is calculated as a percent reduction in absorbance. The percentage of cation radical scavenging was computed using the following equation: Scavenging rate (%) = [A (blank) − A (sample)]/A (blank) × 100

Statistical Analyses
Statistical analyses for DPPH and ABTS cation radical scavenging activities were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). The nonlinear regression was used to determine the dose-response inhibition. Results are expressed as means ± standard deviation of three independent experiments.