Article pubs.acs.org/jnp
Pentacyclic Antibiotics from a Tidal Mud Flat-Derived Actinomycete Kyuho Moon,† Beomkoo Chung,‡ Yoonho Shin,† Arnold L. Rheingold,§ Curtis E. Moore,§ Sung Jean Park,⊥ Sunghyouk Park,† Sang Kook Lee,† Ki-Bong Oh,‡ Jongheon Shin,† and Dong-Chan Oh*,† †
Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea ‡ Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea § Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, United States ⊥ College of Pharmacy, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon 406-799, Republic of Korea S Supporting Information *
ABSTRACT: The combination of investigating a unique source of chemically prolific bacterium with an LC/MS-based bacterial strain selection approach resulted in the discovery of two new secondary metabolites, buanmycin (1) and buanquinone (2), from the culture of a marine Streptomyces strain, which was isolated from a tidal mudflat in Buan, Republic of Korea. The carbon backbone of buanmycin (1), comprising 20 quaternary carbons out of 30 total carbons, was determined via 13C−13C COSY NMR analysis after labeling 1 with 13C by culturing the bacterium with 13C-glucose. The complete structure of 1 was confidently elucidated, primarily based on 1D and 2D NMR spectroscopic and X-ray crystallographic analysis, as that of a new pentacyclic xanthone. The absolute configuration of the α-methyl serine unit in 1 was established by applying the advanced Marfey’s method. The structure of buanquinone (2) was determined to be a new pentacyclic quinone based on NMR and MS spectroscopic data. Buanmycin exhibited potent cytotoxicity against colorectal carcinoma cells (HCT-116) and gastric carcinoma cells (SNU-638) with submicromolar IC50 values and strongly inhibited the pathogenic Gram-negative bacterium Salmonella enterica (MIC = 0.7 μM). In particular, buanmycin demonstrated inhibition of sortase A, which is a promising target for antibiotic discovery.
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actinomycete Salinispora tropica.5 In addition, the application of advanced analytical chemistry technologies to dereplicate previously identified compounds and prioritize structurally new secondary metabolites, rather than using traditional bioassayguided fractionation, could provide more opportunities to discover novel chemicals and thus possibly lead to a new drug discovery paradigm.6 In a natural products discovery program, we have been searching for unique environments that harbor chemically prolific bacteria and have performed LC/MS-based chemical
icrobial secondary metabolites have provided significant benefits to human health by supplying approximately 60−70% of the antibiotics in clinical use.1 Although the history of microbial antibiotics, including the golden era of antibiotics, has been prosperous, the discovery of novel microbial bioactive compounds is currently facing a serious challenge from the redundant isolation of previously reported metabolites.2 Therefore, new search strategies to identify novel microbial secondary metabolites are urgently required. The exploration of microbial sources other than the heavily studied terrestrial microorganisms can be considered as one new approach.3 These efforts have highlighted the potential of marine actinomycetes,4 as represented by Fenical’s discovery of the promising anticancer agent salinosporamide A, which is currently in a phase I clinical trial, from the marine © XXXX American Chemical Society and American Society of Pharmacognosy
Special Issue: Special Issue in Honor of William Fenical Received: September 23, 2014
A
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data (Table 1). The 1H NMR spectrum of 1 in DMSO-d6 revealed the presence of seven broad heteroatom-bound
analysis of these bacterial cultures. The combination of new natural sources and the chemical analysis approach resulted in the isolation of structurally novel and biologically active natural products from a deep-sea actinomycete,7 a volcanic islandderived Streptomyces,8 an Arctic seafloor actinomycete,9 and a rare sponge-symbiotic actinomycete Amycolatopsis sp.10 In the present study, tidal mudflats were investigated, which experience severe changes in temperature, salinity, sunlight, and oxygen availability because these geographic areas are submerged at high tide and exposed at low tide twice daily and thus possibly harbor diverse actinobacteria.11 It was hypothesized that there are actinobacterial communities that may be able to biosynthesize new bioactive secondary metabolites. Therefore, bacterial strains were isolated from a tidal mudflat in Buan, Republic of Korea, which is designated as a Ramsar wetland. Analysis of the secondary metabolites was performed on these bacterial strains by LC/MS to prioritize those producing new chemistry. During the chemical screening process, it was found that the strain SNR69, belonging to the genus Streptomyces, produces a major compound that displays a distinctive UV spectrum (λmax 297 nm) and a molecular ion peak at m/z 594. The initial dereplication, based on the UV and MS data, indicated clearly that the major compound was probably unknown and served as a guide to study the chemistry of the strain further. Reported here are the structures of the major compound, buanmycin (1), and a minor metabolite, buanquinone (2). The biological activities of these new compounds were evaluated in bacterial sortase A inhibition, cytotoxicity, and antimicrobial activity assays.
Table 1. 1H and 13C NMR Data for Buanmycin (1) in DMSO-d6a position
a1
δC, type
1 2 2a 2b
174.2, 60.3, 19.7, 65.5,
C C CH3 CH2
4 5 6 6a 6b 7 7a 8 8a 9 10 11 12 12a 13a 14 14a 15 16 16a 17 18 19
166.8, 126.9, 147.3, 118.4, 114.5, 148.8, 105.9, 185.8, 107.6, 149.0, 142.7, 122.0, 106.2, 148.8, 144.6, 138.3, 133.7, 23.6, 29.3, 140.1, 121.3, 133.1, 46.7,
C C C C C C C C C C C CH CH C C C C CH2 CH2 C CH C CH2
20 21 OH-1 OH-2b OH-6 OH-7 OH-9 OCH3-10 exchangeable H exchangeable H
205.8, C 29.7, CH3
56.7, CH3
δH, mult. (J in Hz)
1.44, s 3.68, d (11.0) 3.61, d (11.0)
7.61, d (9.0) 7.11, d (9.0)
2.80, br m 2.64, br m 6.71, s 3.84, d (16.0) 3.78, d (16.0) 2.14, s 13.06, s 4.84, br m 12.46, s 11.86, br s 11.84, br s 3.87, s 9.46, s 8.06, s
H and 13C data were recorded at 600 and 125 MHz, respectively.
protons (δH 13.06, 12.46, 11.86, 11.84, 9.46, 8.06, 4.84), two ortho-coupled aromatic protons [δH 7.61 (d, J = 9.0 Hz); 7.11 (d, J = 9.0 Hz)], one singlet aromatic proton (δH 6.71), one methoxy group (δH 3.87), eight aliphatic protons [δH 3.84, 3.78, 3.68, 3.61, 2.80 (2H), 2.64 (2H)], and two singlet methyl groups (δH 2.14, 1.44). The 13C NMR data displayed signals for four carbonyl groups (δC 205.8, 185.8, 174.2, 166.8), 18 carbon signals between δC 149.0 and 105.9, indicating a polyunsaturated structure, and eight sp3 carbon signals between δC 65.5 and 19.7. The analysis of the HSQC NMR spectrum allowed the assignment of all the carbon-bound protons to their corresponding carbons, identifying three methyl groups, including one methoxy carbon (δC 56.7), four methylenes, three methines, and 20 quaternary carbons. Since the structure of 1 contains 20 quaternary carbons out of 30 total carbons, the
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RESULTS AND DISCUSSION Buanmycin (1) was isolated as a yellow powder. The molecular formula of 1 was assigned as C30H27NO12 based on the HRFAB mass spectrometric data (m/z 594.1613 [M + H]+, calcd for C30H28NO12, 594.1612 [M + H]+) and the 1H and 13C NMR B
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1
ring. Despite all of the observed HMBC correlations, some connectivities remained unassigned. The positions of OH-6 and OH-14, the ether linkage forming a xanthone ring, and the location of the α-methyl serine were finally determined by Xray crystallographic analysis (Figure 3). The results indicated
H-based detection NMR experiments provided only limited insight into the full structure of 1. Therefore, the acquisition of the 13C-based NMR spectrum was considered, but the low abundance of 13C required labeling 1 with 13C. The strain was enriched with 13C by feeding the bacterial cultures with uniformly labeled 13C-glucose to produce 13C-enriched buanmycin (1), and the incorporation of 13C into the molecule was confirmed with LC/MS analysis. After purifying the labeled compound, 13C−13C COSY NMR spectroscopy was performed.12−15 From the 13C−13C COSY NMR spectroscopy results, the connectivity between two adjacent carbons could be elucidated by direct 13C−13C coupling. The analysis of the observed 13C−13C COSY correlations clearly established the majority of the carbon backbone connectivity of 1, except for the C-5/C-6, C-7/C-7a, and C-10/C-11 correlations (Figure 1;
Figure 3. ORTEP diagram for buanmycin (1).
the new pentacyclic xanthone structure of buanmycin (1). The absolute configuration of the only stereogenic center, C-2 in the α-methyl serine, was determined as S by acid hydrolysis and applying the advanced Marfey’s method.16,17 Buanquinone (2) was isolated as a red powder. The molecular formula of 2 was assigned as C25H18O7 based on the 1H and 13C NMR (Table 2) spectra and the HRFAB mass spectrometric data (m/z 431.1139 [M + H]+, calcd for C25H19O7, 431.1131 [M + H]+). The 1H NMR spectrum of 2 in DMSO-d6 displayed signals for four phenolic hydroxy groups (δH 12.55, 12.15, 11.49, 10.39), two meta-coupled aromatic protons [δH 7.17 (d, J = 2.0 Hz), 6.62 (d, J = 2.0 Hz)], three aromatic singlet protons (δH 8.83, 6.73, 6.65), one singlet methyl group (δH 2.16), and six aliphatic protons [δH 3.71 (2H), 2.83 (2H), 2.73 (2H)]. The 13C NMR and HSQC spectra revealed three signals between δC 205.8 and 181.7, suggesting the presence of three conjugated carbonyl groups and 18 carbons between δC 165.6 and 107.9, similar to the carbon signal pattern of buanmycin (1), suggesting a polyaromatic system. Detailed one-dimensional (1D) and two-dimensional (2D) NMR spectroscopic analysis, including 1 H−1H COSY, HSQC, and HMBC measurements, was used to determine the structure of 2 (Figure 2). The HMBC correlations of the two methine protons (H-14, H-12) and two phenolic hydroxy protons (OH-9, OH-7) supported the elucidation of the anthraquinone moiety. The methine proton H-12 (δH 7.17) exhibited HMBC correlations to C-13 (δC 181.7), C-11 (δC 165.6), C-8a (δC 109.3), and C-10 (δC 107.9) and correlated with H-10 (δH 6.62) through meta coupling. HMBC correlations were observed from OH-9 (δH 12.15) to C-10 (δC 107.9) and C-8a (δC 109.3). Long-range heteronuclear coupling from OH-7 (δH 12.55) to C-7 (δC 158.0), C7a (δC 113.0), and C-6a (δC 132.3) was used to locate the hydroxy group at C-7. The downfield proton of H-14 (δH 8.83), which correlated with C-13 (δC 181.7), C-7a (δC 113.0), and C-6a (δC 132.3), established the anthraquinone moiety as one that incorporates two ketones at C-8 (δC 189.8) and C-13. The methylene signal protons H2-6 (δH 2.83) exhibited HMBC correlations to C-7 (δC 158.0) and C-6a (δC 132.3), connecting
Figure 1. Observed 13C−13C COSY correlations elucidating the carbon backbone of buanmycin (1).
see also the Supporting Information, Figure S6). Further analysis of the 1H−1H COSY and HMBC correlations was used to refine the backbone structure of 1 (Figure 2). The analysis of
Figure 2. Key 1H−1H COSY and HMBC correlations of 1 and 2.
the COSY NMR spectrum helped define the connectivity between the two methylenes (C-14 and C-15) and the orthocoupled aromatic methines (C-11 and C-12). The α-methyl serine unit was confirmed based on the HMBC correlations from the protons of the C-2a methyl group [H3-2a (δH 1.44)] to C-2 (δC 60.3), C-2b (δC 65.5), and C-1 (δC 174.2)]. This unit was established further by the HMBC coupling from H-2b to C-2, C-2a, and C-1. The long-range 1H−13C NMR correlations from H3-21 (δH 2.14) to the C-20 carbonyl (δC 205.8) and C-19 (δC 46.7) and from H2-19 to C-20 and C-18 (δC 133.1) confirmed the location of the C3 side chain at the left-end aromatic ring. The two-bond and three-bond HMBC correlations originating from the phenolic hydroxy groups (OH-7 and OH-9) determined their locations at C-7 and C-9, respectively. The methoxy signal was assigned to C-10 by the HMBC cross-peak between OCH3-10 (δH 3.87) and C-10 (δC 142.7). The heteronuclear correlations from H-11 and H-12 to C-9 and C-10, respectively, completed the right-end aromatic C
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Table 2. 1H and 13C NMR Data for Buanquinone (2) in DMSO-d6a position 1 2 3 4 4a 5 6 6a 7 7a 8 8a 9 10 11 12 12a 13 13a 14 14a 14b 15 16 17 OH-1 OH-7 OH-9 OH-11 a1
δC, type 155.9, 116.4, 137.3, 120.5, 140.3, 28.1, 20.5, 132.3, 158.0, 113.0, 189.8, 109.3, 164.5, 107.9, 165.6, 108.8, 135.5, 181.7, 130.2, 119.9, 141.0, 118.4, 49.6, 205.8, 29.7,
C CH C CH C CH2 CH2 C C C C C C CH C CH C C C CH C C CH2 C CH3
Table 3. Cytotoxicity Data IC50 for 1 and 2 against Selected Cancer Cell Lines
δH, mult. (J in Hz)
carcinoma
A549
HCT116
SNU638
K562
SKHEP1
MDA-MB231
1 2 etoposide
1.7 − 0.8
0.9 − 1.9
0.8 − 0.5
−a 18.3 3.1
1.9 − 1.1
1.2 − 10.6
6.73, s 6.65, s
a
2.73, m 2.83, m
IC50 > 100 μM.
the K562 cells. Buanquinone (2) exhibited moderate cytotoxicity against the K562 cell line but did not display any significant cytotoxicity against the other cell lines tested (Table 3). The antibacterial activities of the citreamicins against Grampositive bacteria have been reported previously. Therefore, the antibacterial activities of 1 and 2 were evaluated against Grampositive bacteria, including Staphylococcus aureus, Bacillus subtilis, and Kocuria rhizophila, as well as the Gram-negative pathogens Salmonella enterica and Proteus hauseri (Table 4).
6.62, d (2.0) 7.17, d (2.0)
Table 4. Antibacterial Activity Data for 1 and 2 MIC (μM) Gram-positive
8.83, s
3.71, s 2.16 10.39, 12.55, 12.15, 11.49,
a
s s s br s
Gram-negative
compound
S. aureus
B. subtilis
K. rhizophila
S. enterica
P. hauseri
1 2 ampicillin
10.5 −a 4.5
0.7 − 1.1
10.5 − 0.6
0.7 − 4.5
21.1 − 100 μg/mL.
The activity profile of 1 revealed that buanmycin is notably potent against the Gram-negative bacterium S. enterica, which causes salmonellosis. Buanquinone (2) did not demonstrate significant antibacterial activity. Additionally, buanmycin (1) exhibited moderate antifungal activity against C. albicans [minimum inhibitory concentration (MIC) = 21.1 μM]. In the antifungal assay against filamentous fungi, only buanmycin (1) displayed weak antifungal activity against A. f umigatus, with a MIC value of 84.3 μM. However, buanquinone did not exhibit significant antifungal activity. Since buanmycin (1) inhibited the growth of pathogenic bacteria, its biological activity was evaluated further in a Staphylococcus aureus sortase A inhibition assay. Sortase A is an enzyme from the transpeptidase family that plays a key role in adhesion and host invasion by Gram-positive bacteria.21 Notably, buanmycin inhibited Staphylococcus aureus sortase A and, with an IC50 value of 43.2 μM, was more potent than the positive control compound, p-hydroxymercuribenzoic acid (pHMB) (IC50: 104.4 μM).
H and 13C data were recorded at 600 and 125 MHz, respectively.
C-6 (δC 20.5) to C-6a. The observation of the COSY correlation between H2-6 and H-5 (δH 2.73) allowed the assignment of C-5 (δC 28.1) next to C-6. The HMBC correlations from H-5 to C-14b (δC 118.4), C-4a (δC 140.3), and C-4 (δC 120.5), from H-4 (δH 6.65) to C-4a (δC 140.3), and from OH-1 (δH 10.39) to C-14b (δC 118.4) indicated the entire cyclic system. In turn, the aliphatic chains that were linked to C-3 (δC 137.3) were indicated to have one methylene (δC 49.6-δH 3.71), one methyl group (δC 29.7-δH 2.16), and a carbonyl group (δC 205.8) by their HMBC correlations. Thus, the full structure of buanquinone (2) was completely assigned as a new pentacyclic anthraquinone. Even though buanmycin (1) shares a common xanthone feature with the citreamicins, including citreamicin ε A−B18 and citreamicin θ A−B,19 the pentacyclic (6/6/6/6/6) structure of 1 differs from the septacyclic (5/6/6/6/6/6/6) system in every citreamicin class of antibiotics. Buanquinone (2) is structurally related to KS-191-1, which originates from Streptomyces californicus,20 but lacks one carboxylic acid unit at C-2. The biological activities of 1 and 2 were evaluated in several bioassays. The cytotoxicities of 1 and 2 were tested against the following human carcinoma cell lines: A549 (lung cancer), HCT116 (colon cancer), SNU638 (gastric cancer), K562 (leukemia), SK-HEP1 (liver cancer), and MDA-MB231 (breast cancer) (Table 3). Buanmycin (1) exhibited potent cytotoxicity against the A549, HCT116, SNU638, SK-HEP1, and MDAMB-231 cells, with IC50 values of 0.8 to 1.9 μM, but not against
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EXPERIMENTAL SECTION
General Experimental Procedures. The optical rotations were measured with a JASCO P-200 polarimeter. The UV spectra were recorded on a PerkinElmer Lambda 35 UV/vis spectrometer. The IR spectra were acquired with a Thermo Nicolet iS10 spectrometer. The 1 H, 13C, and 2D NMR spectra were recorded on Bruker Avance 600 MHz spectrometers at the National Center for Interuniversity Research Facilities at Seoul National University (NCIRF) and at the Gachon University. The low-resolution electrospray ionization source mass spectra were obtained with an Agilent Technologies 6130 quadrupole mass spectrometer coupled to an Agilent Technologies 1200 series high-performance liquid chromatography (HPLC) instrument. High-resolution fast-atom bombardment (HRFAB) mass spectra were obtained using a JEOL JMS-600W high-resolution D
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optics. A total of 14 404 reflections, collected in the θ range 2.48° to 68.27°, yielded 8792 unique reflections (Rint = 0.0175), final R indices [I > 2σ(I)] R1 = 0.0327, wR2 = 0.0882. The structure was solved by direct methods (SHELXS-97) and refined by full-matrix least-squares on F2 (goodness of fit on F2 = 1. 060). Carbon, oxygen, and nitrogen atoms were refined anisotropically. Hydrogen atoms were placed in idealized locations except for two hydroxyl H atoms, which were refined freely with isotropic displacement parameters. The absolute stereochemistry was unambiguously determined as shown by the value of the refined Flack parameter. The asymmetric unit consists of two crystallographically independent, but chemically similar molecules. Absorption correction was performed by SADABS using with multiscan parameters. Crystallographic data for 1 have been deposited at the Cambridge Crystallographic Data Centre (CCDC 1018479). Copies of these data can be obtained free of charge via the Internet at www.ccdc.cam.ac.uk/conts/retrieving.html. Crystal data of buanmycin (1): C34H35NO14, M = 681.63, triclinic crystal system, crystal size 0.27 × 0.21 × 0.08 mm3, space group P1, a = 7.4957(3) Å, b = 12.0298(4) Å, c = 17.8347(7) Å, α = 87.3870(12)°, β = 87.5550(11)°, γ = 74.7080(10)°, V = 1548.85(10) Å3, Z = 2, μ = 0.970 mm−1, Dc = 1.462 g/cm3. F(000) = 716, Flack parameter x = −0.02(12). Cytotoxicity Assay. The cytotoxicity for cultured cancer cells of compounds 1 and 2 was evaluated with a sulforhodamine B (SRB) assay. Six human cancer cell lines (HCT116, MDA-MB231, SNU638, A549, K562, and SK-HEP1) were seeded into 96-well plates at 3 × 104 cells/mL and incubated with various concentrations of each compound at 37 °C in a humidified atmosphere with 5% CO2. After 72 h, the cells were fixed with a 10% TCA solution for 1 h, and the cellular proteins were stained with 0.4% SRB in 1% acetic acid. The stained cells were dissolved in 10 mM Tris buffer (pH 10.0). The effects of 1 and 2 on cell cytotoxicity were calculated as a percentage relative to the solvent-treated control. The IC50 values were determined by nonlinear regression analysis (percent survival versus concentration). Etoposide was used as the positive control. Antibacterial Activity Assay. Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, and Kocuria rhizophila NBRC 12708) and Gram-negative bacteria (Salmonella enterica ATCC 14028, Proteus hauseri NBRC 3851, and Escherichia coli ATCC 25922) were used for the antimicrobial activity tests.22 The bacteria were incubated overnight in Luria−Bertani (LB) broth at 37 °C, harvested by centrifugation, and washed twice with sterile distilled water. The compound solutions were prepared in DMSO. Each solution was diluted with Plate Count Broth (Difco) to generate serial 2-fold dilutions in the range of 50 to 0.8 μg/mL. Aliquots (10 μL) of the broth containing the test bacteria were added to each 96-well microtiter plate at a final concentration of 5 × 105 colony-forming units (cfu)/mL. The plates were incubated for 12 h at 37 °C. The MIC values were determined as the lowest concentration at which there is no visible growth. Ampicillin was used as a reference compound. Antifungal Activity Assay. Candida albicans ATCC 10231 was cultivated with YPD medium (1% yeast extract, 2% peptone, and 2% dextrose). After incubation for 48 h at 28 °C, the cells were harvested by centrifugation and washed twice with sterile distilled water. Aspergillus f umigatus HIC 6094, Trichophyton rubrum NBRC 9185, and T. mentagrophytes IFM 40996 were plated onto potato dextrose agar plates and incubated for 2 weeks at 28 °C. Spores were harvested and washed twice with sterile water, and then the spores were resuspended in water to an initial inoculum concentration of 5 × 105 spores/mL. In each well of a 96-well plate, 10 μL of the cell suspension was mixed with 90 μL of potato dextrose broth (Difco) (5 × 104 cells/mL) containing the test compound in DMSO solutions (final concentration, 0.5%).22 A culture containing DMSO (0.5%) and a culture supplemented with amphotericin B were used as a solvent control and as a positive control, respectively. Sortase A Inhibition Assay. Staphylococcus aureus ATCC 6538p was the source of the srtA gene. The expression and purification of recombinant SrtAΔ24 were carried out according to a previously published procedure.23 The reactions were performed in 100 μL
mass spectrometer at the NCIRF. The semipreparative HPLC separations were performed with a Gilson 305 pump and a Gilson UV/vis-155 detector. Bacterial Isolation and Cultivation. The sample was collected in June 2011 from a tidal mud flat in Buan, Republic of Korea. Dried sediment was pressed onto the surface of actinomycete isolation medium (1 L of seawater, 18 g of agar, 100 mg/L cycloheximide) and A5 (750 mL of seawater, 250 mL of distilled water, 18 g of agar, 100 mg/L cycloheximide), A6 (1 L of seawater, 18 g of agar, and 5 mg/L polymyxin B sulfate), and A7 (1 L of seawater, 18 g of agar, and 5 mg/ L kanamycin) media agar plates using an autoclaved foam plug (2 cm in diameter). Then, 50 μL of the suspension, the mixture of 0.5 g of dried sediment and 10 mL of sterilized artificial seawater, was heated to 55 °C and inoculated onto the agar plates. The plates were incubated at 25 °C for 3 weeks. The strain SNR69 was isolated from the actinomycete isolation medium plates. The sequence analysis of the 16S rDNA sequencing analysis data (GenBank accession number: KM353585) obtained from COSMO Co., Ltd. revealed that strain SNR69 was most similar to Streptomyces cyaneus (99% identity). SNR69 was cultivated in 1 L of YEME medium (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, and 27 g of artificial sea salt) at 170 rpm and 30 °C for 5 days. Altogether 20 L of bacterial liquid culture was prepared. Extraction and Isolation. The entire culture was extracted twice with 30 L of ethyl acetate. The EtOAc extract was concentrated in vacuo to yield 7 g of dry material. One-half of the dried extract was fractionated with 150 mL each of 20%, 40%, 60%, 80%, and 100% MeOH in water using a packed C18 (20 g) column. After the fractionation, buanmycin (1) was identified in the 60% and 80% fractions, and buanquinone (2) was detected in the 80% and 100% fractions. Each fraction was then subjected to reversed-phase HPLC (Kromasil 100-5-C18 250 × 10 mm, flow rate 2 mL/min, UV 280 nm detection, with a 50−60% aqueous acetonitrile gradient, 0.1% formic acid, over 60 min). Under these purification conditions, buanmycin (1) and buanquinone (2) eluted at 10 and 51 min, respectively. Finally, the procedures were repeated to afford pure buanmycin (1) (22 mg) and buanquinone (2) (6 mg). Buanmycin (1): yellow powder; [α]25D +72 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 216 (2.35), 239 (2.21), 276 (2.33), 297 (2.64), 424 (0.28) nm; IR (neat) νmax 3361, 2851, 1706, 1630, 1491, 1282, 1038 cm−1; 1H and 13C NMR data, see Table 1; HRFABMS m/z 594.1613 [M + H]+ (calcd for C30H28NO12, 594.1612). Buanquinone (2): red powder; [α]25D +128 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 206 (1.20), 309 (0.89), 473 (0.40) nm; IR (neat) νmax 3841, 3394, 2928, 1681, 1442, 1208, 1140 cm−1; 1H and 13 C NMR data, see Table 2; HRFABMS m/z 431.1139 [M + H]+ (calcd for C25H19O7, 431.1131). 13 C Labeling of Buanmycin (1). The bacterial strain SNR69 was cultivated in 50 mL of YEME medium. After 3 days on a rotary shaker at 170 rpm and 30 °C, 10 mL of the culture was inoculated into 200 mL of a 13C-enriched medium (0.8 g of uniformly labeled 13C-glucose, 0.8 g of yeast extract, 2 g of malt extract, and 5.4 g of artificial sea salt). In total, 3.2 L (16 each × 200 mL) was cultivated for 5 days. The 13Cenriched buanmycin was purified as described above. 13 C−13C COSY NMR Spectroscopy. The 13C−13C COSY NMR experiment was performed in a constant-time fashion to remove the vicinal carbon−carbon splitting in the t1 dimension. The 180° inversion of the carbon nuclei was achieved using an adiabatic pulse for broadband applications (34722 Hz). The proton spins were decoupled using a WALTZ-65 supercycle during the entire pulse sequence, except for a recycle delay of 2 s. The total number of points was 1024 (t2) × 256 (t1) complex points, and the spectral widths for both dimensions were 230 ppm. The entire experiment required 2 days and 8 h with 9 mg of buanmycin (1). Crystallization and X-ray Crystallographic Analysis of Buanmycin (1). Buanmycin was crystallized by slow evaporation of a solution in EtOAc−hexane. The crystal structure and absolute configuration of 1 were determined using data collected at T = 100(2) K with Cu Kα radiation (λ = 1.54178 Å) on a Bruker D8 diffractometer with an FR-591 rotating-anode source and microfocus E
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containing 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, 5 mM CaCl2, 1 μg of fluorescent peptide (H2N-Abz-LPXTG-Dap(Dnp)-NH2), 1 mM pentaglycine, 24 μg of recombinant SrtAΔ24, and the test compounds at various concentrations (0.5% DMSO final concentration). Appropriate blanks contained all of the above constituents, with the exception of the test sample. The reactions were performed for 1 h at 37 °C. The sample fluorescence was measured using emission and excitation wavelengths of 420 and 317 nm, respectively. pHydroxymercuribenzoic acid, a known sortase inhibitor,24 was used as a positive control.
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(16) (a) Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem. 1997, 69, 3346−3352. (b) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem. 1997, 69, 5146−5151. (17) Miller, E. D.; Kauffman, C. A.; Jensen, P. R.; Fenical, W. J. Org. Chem. 2007, 72, 323−330. (18) (a) Carter, G. T.; Nietsche, J. A.; Williams, D. R.; Borders, D. B. J. Antibiot. 1990, 43, 504−512. (b) Liu, L.-L.; He, L.-S.; Xu, Y.; Han, Z.; Li, Y.-X.; Zhong, J.-L.; Guo, X.-R.; Zhang, X.-X.; Ko, K. M.; Qian, P.-Y. Chem. Res. Toxicol. 2013, 26, 1055−1063. (19) Liu, L.-L.; Xu, Y.; Han, Z.; Li, Y.-X.; Lu, L.; Lai, P.-Y.; Zhong, J.L.; Guo, X.-R.; Zhang, X.-X.; Qian, P.-Y. Mar. Drugs 2012, 10, 2571− 2583. (20) (a) Matsuda, Y.; Kase, H. J. Antibiot. 1987, 40, 1104−1110. (b) Yasuzawa, T.; Yoshida, M.; Shirahata, K.; Sano, H. J. Antibiot. 1987, 40, 1111−1114. (21) Hendrickx, A. P.; Budzik, J. M.; Oh, S.-Y.; Schneewind, O. Nat. Rev. Microbiol. 2011, 9, 166−176. (22) Oh, K.-B.; Lee, J. H.; Chung, S.-C.; Shin, J.; Shin, H. J.; Kim, H.K.; Lee, H.-S. Bioorg. Med. Chem. Lett. 2008, 18, 104−108. (23) Oh, K.-B.; Kim, S.-H.; Lee, J.; Cho, W.-J.; Lee, T.; Kim, S. J. Med. Chem. 2004, 47, 2418−2421. (24) Ton-That, H.; Liu, G.; Mazmanian, S. K.; Schneewind, O. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12424−12429.
ASSOCIATED CONTENT
S Supporting Information *
The detailed experimental procedures and NMR data for compounds 1 and 2 and X-ray crystallographic data of 1 are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(D.-C. Oh) Tel: +82-2-880-2491. Fax: +82-2-762-8322. Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Prof. C. C. Hughes, University of California, San Diego, for assistance with the X-ray crystallographic analysis. This work was supported by National Research Foundation of Korea grants funded by the Korean Government (Ministry of Science, ICT and Future Planning) (M1A5A1-2010-0020429 and 2009-0083533).
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DEDICATION Dedicated to Dr. William Fenical of Scripps Institution of Oceanography, University of California−San Diego, for his pioneering work on bioactive natural products.
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REFERENCES
(1) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311−335. (2) Ortholand, J.-Y.; Ganesan, A. Curr. Opin. Chem. Biol. 2004, 8, 271−280. (3) Clardy, J.; Fischbach, M. A. Wiley Encyclopedia of Chemical Biology; John Wiley and Sons, 2009; Vol. 3, pp 240−249. (4) Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666−673. (5) Fenical, W.; Jensen, P. R.; Palladino, M. A.; Lam, K. S.; Lloyd, G. K.; Potts, B. C. Bioorg. Med. Chem. 2009, 17, 2175−2180. (6) Krug, D.; Müller, R. Nat. Prod. Rep. 2014, 31, 768−783. (7) Um, S.; Kim, Y.-J.; Kwon, H.; Wen, H.; Kim, S.-H.; Kwon, C. H.; Park, S.; Shin, J.; Oh, D.-C. J. Nat. Prod. 2013, 76, 873−879. (8) Um, S.; Choi, T. J.; Kim, H.; Kim, B. Y.; Kim, S.-K.; Lee, S. K.; Oh, K.-B.; Shin, J.; Oh, D.-C. J. Org. Chem. 2013, 78, 12321−12329. (9) Moon, K.; Ahn, C.-H.; Shin, Y.; Won, T. H.; Ko, K.; Lee, S. K.; Oh, K.-B.; Shin, J.; Nam, S.-I.; Oh, D.-C. Mar. Drugs 2014, 12, 2526− 2538. (10) Kwon, Y.; Kim, S.-H.; Shin, Y.; Bae, M.; Kim, B. Y.; Lee, S. K.; Oh, K.-B.; Shin, J.; Oh, D.-C. Mar. Drugs 2014, 12, 2326−2340. (11) Stevens, H.; Brinkhoff, T.; Rink, B.; Vollmers, J.; Simon, M. Environ. Microbiol. 2007, 9, 1810−1822. (12) Kawabata, J.; Fukushi, E. J. Magn. Reson. 1995, 117, 88−90. (13) Reif, B.; Kock, M.; Kerssebaum, R.; Kang, H.; Fenical, W.; Griesinger, C. J. Magn. Reson. 1996, 118, 282−285. (14) Uhrín, D. Annu. Rep. NMR Spectrosc. 2010, 70, 1−34. (15) Kwon, Y.; Park, S.; Shin, J.; Oh, D.-C. Arch. Pharm. Res. 2014, 37, 967−971. F
dx.doi.org/10.1021/np500736b | J. Nat. Prod. XXXX, XXX, XXX−XXX
Pentacyclic Antibiotics from a Tidal Mud Flat-Derived Actinomycete Kyuho Moon,† Beomkoo Chung,‡ Yoonho Shin,† Arnold L. Rheingold,§ Curtis E. Moore,§ Sung Jean Park,⊥ Sunghyouk Park,† Sang Kook Lee,† Ki-Bong Oh,‡ Jongheon Shin,† and Dong-Chan Oh*,† †
Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea ‡ Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea § Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0358, United States ⊥ College of Pharmacy, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon 406-799, Republic of Korea S Supporting Information *
ABSTRACT: The combination of investigating a unique source of chemically prolific bacterium with an LC/MS-based bacterial strain selection approach resulted in the discovery of two new secondary metabolites, buanmycin (1) and buanquinone (2), from the culture of a marine Streptomyces strain, which was isolated from a tidal mudflat in Buan, Republic of Korea. The carbon backbone of buanmycin (1), comprising 20 quaternary carbons out of 30 total carbons, was determined via 13C−13C COSY NMR analysis after labeling 1 with 13C by culturing the bacterium with 13C-glucose. The complete structure of 1 was confidently elucidated, primarily based on 1D and 2D NMR spectroscopic and X-ray crystallographic analysis, as that of a new pentacyclic xanthone. The absolute configuration of the α-methyl serine unit in 1 was established by applying the advanced Marfey’s method. The structure of buanquinone (2) was determined to be a new pentacyclic quinone based on NMR and MS spectroscopic data. Buanmycin exhibited potent cytotoxicity against colorectal carcinoma cells (HCT-116) and gastric carcinoma cells (SNU-638) with submicromolar IC50 values and strongly inhibited the pathogenic Gram-negative bacterium Salmonella enterica (MIC = 0.7 μM). In particular, buanmycin demonstrated inhibition of sortase A, which is a promising target for antibiotic discovery.
M
actinomycete Salinispora tropica.5 In addition, the application of advanced analytical chemistry technologies to dereplicate previously identified compounds and prioritize structurally new secondary metabolites, rather than using traditional bioassayguided fractionation, could provide more opportunities to discover novel chemicals and thus possibly lead to a new drug discovery paradigm.6 In a natural products discovery program, we have been searching for unique environments that harbor chemically prolific bacteria and have performed LC/MS-based chemical
icrobial secondary metabolites have provided significant benefits to human health by supplying approximately 60−70% of the antibiotics in clinical use.1 Although the history of microbial antibiotics, including the golden era of antibiotics, has been prosperous, the discovery of novel microbial bioactive compounds is currently facing a serious challenge from the redundant isolation of previously reported metabolites.2 Therefore, new search strategies to identify novel microbial secondary metabolites are urgently required. The exploration of microbial sources other than the heavily studied terrestrial microorganisms can be considered as one new approach.3 These efforts have highlighted the potential of marine actinomycetes,4 as represented by Fenical’s discovery of the promising anticancer agent salinosporamide A, which is currently in a phase I clinical trial, from the marine © XXXX American Chemical Society and American Society of Pharmacognosy
Special Issue: Special Issue in Honor of William Fenical Received: September 23, 2014
A
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data (Table 1). The 1H NMR spectrum of 1 in DMSO-d6 revealed the presence of seven broad heteroatom-bound
analysis of these bacterial cultures. The combination of new natural sources and the chemical analysis approach resulted in the isolation of structurally novel and biologically active natural products from a deep-sea actinomycete,7 a volcanic islandderived Streptomyces,8 an Arctic seafloor actinomycete,9 and a rare sponge-symbiotic actinomycete Amycolatopsis sp.10 In the present study, tidal mudflats were investigated, which experience severe changes in temperature, salinity, sunlight, and oxygen availability because these geographic areas are submerged at high tide and exposed at low tide twice daily and thus possibly harbor diverse actinobacteria.11 It was hypothesized that there are actinobacterial communities that may be able to biosynthesize new bioactive secondary metabolites. Therefore, bacterial strains were isolated from a tidal mudflat in Buan, Republic of Korea, which is designated as a Ramsar wetland. Analysis of the secondary metabolites was performed on these bacterial strains by LC/MS to prioritize those producing new chemistry. During the chemical screening process, it was found that the strain SNR69, belonging to the genus Streptomyces, produces a major compound that displays a distinctive UV spectrum (λmax 297 nm) and a molecular ion peak at m/z 594. The initial dereplication, based on the UV and MS data, indicated clearly that the major compound was probably unknown and served as a guide to study the chemistry of the strain further. Reported here are the structures of the major compound, buanmycin (1), and a minor metabolite, buanquinone (2). The biological activities of these new compounds were evaluated in bacterial sortase A inhibition, cytotoxicity, and antimicrobial activity assays.
Table 1. 1H and 13C NMR Data for Buanmycin (1) in DMSO-d6a position
a1
δC, type
1 2 2a 2b
174.2, 60.3, 19.7, 65.5,
C C CH3 CH2
4 5 6 6a 6b 7 7a 8 8a 9 10 11 12 12a 13a 14 14a 15 16 16a 17 18 19
166.8, 126.9, 147.3, 118.4, 114.5, 148.8, 105.9, 185.8, 107.6, 149.0, 142.7, 122.0, 106.2, 148.8, 144.6, 138.3, 133.7, 23.6, 29.3, 140.1, 121.3, 133.1, 46.7,
C C C C C C C C C C C CH CH C C C C CH2 CH2 C CH C CH2
20 21 OH-1 OH-2b OH-6 OH-7 OH-9 OCH3-10 exchangeable H exchangeable H
205.8, C 29.7, CH3
56.7, CH3
δH, mult. (J in Hz)
1.44, s 3.68, d (11.0) 3.61, d (11.0)
7.61, d (9.0) 7.11, d (9.0)
2.80, br m 2.64, br m 6.71, s 3.84, d (16.0) 3.78, d (16.0) 2.14, s 13.06, s 4.84, br m 12.46, s 11.86, br s 11.84, br s 3.87, s 9.46, s 8.06, s
H and 13C data were recorded at 600 and 125 MHz, respectively.
protons (δH 13.06, 12.46, 11.86, 11.84, 9.46, 8.06, 4.84), two ortho-coupled aromatic protons [δH 7.61 (d, J = 9.0 Hz); 7.11 (d, J = 9.0 Hz)], one singlet aromatic proton (δH 6.71), one methoxy group (δH 3.87), eight aliphatic protons [δH 3.84, 3.78, 3.68, 3.61, 2.80 (2H), 2.64 (2H)], and two singlet methyl groups (δH 2.14, 1.44). The 13C NMR data displayed signals for four carbonyl groups (δC 205.8, 185.8, 174.2, 166.8), 18 carbon signals between δC 149.0 and 105.9, indicating a polyunsaturated structure, and eight sp3 carbon signals between δC 65.5 and 19.7. The analysis of the HSQC NMR spectrum allowed the assignment of all the carbon-bound protons to their corresponding carbons, identifying three methyl groups, including one methoxy carbon (δC 56.7), four methylenes, three methines, and 20 quaternary carbons. Since the structure of 1 contains 20 quaternary carbons out of 30 total carbons, the
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RESULTS AND DISCUSSION Buanmycin (1) was isolated as a yellow powder. The molecular formula of 1 was assigned as C30H27NO12 based on the HRFAB mass spectrometric data (m/z 594.1613 [M + H]+, calcd for C30H28NO12, 594.1612 [M + H]+) and the 1H and 13C NMR B
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1
ring. Despite all of the observed HMBC correlations, some connectivities remained unassigned. The positions of OH-6 and OH-14, the ether linkage forming a xanthone ring, and the location of the α-methyl serine were finally determined by Xray crystallographic analysis (Figure 3). The results indicated
H-based detection NMR experiments provided only limited insight into the full structure of 1. Therefore, the acquisition of the 13C-based NMR spectrum was considered, but the low abundance of 13C required labeling 1 with 13C. The strain was enriched with 13C by feeding the bacterial cultures with uniformly labeled 13C-glucose to produce 13C-enriched buanmycin (1), and the incorporation of 13C into the molecule was confirmed with LC/MS analysis. After purifying the labeled compound, 13C−13C COSY NMR spectroscopy was performed.12−15 From the 13C−13C COSY NMR spectroscopy results, the connectivity between two adjacent carbons could be elucidated by direct 13C−13C coupling. The analysis of the observed 13C−13C COSY correlations clearly established the majority of the carbon backbone connectivity of 1, except for the C-5/C-6, C-7/C-7a, and C-10/C-11 correlations (Figure 1;
Figure 3. ORTEP diagram for buanmycin (1).
the new pentacyclic xanthone structure of buanmycin (1). The absolute configuration of the only stereogenic center, C-2 in the α-methyl serine, was determined as S by acid hydrolysis and applying the advanced Marfey’s method.16,17 Buanquinone (2) was isolated as a red powder. The molecular formula of 2 was assigned as C25H18O7 based on the 1H and 13C NMR (Table 2) spectra and the HRFAB mass spectrometric data (m/z 431.1139 [M + H]+, calcd for C25H19O7, 431.1131 [M + H]+). The 1H NMR spectrum of 2 in DMSO-d6 displayed signals for four phenolic hydroxy groups (δH 12.55, 12.15, 11.49, 10.39), two meta-coupled aromatic protons [δH 7.17 (d, J = 2.0 Hz), 6.62 (d, J = 2.0 Hz)], three aromatic singlet protons (δH 8.83, 6.73, 6.65), one singlet methyl group (δH 2.16), and six aliphatic protons [δH 3.71 (2H), 2.83 (2H), 2.73 (2H)]. The 13C NMR and HSQC spectra revealed three signals between δC 205.8 and 181.7, suggesting the presence of three conjugated carbonyl groups and 18 carbons between δC 165.6 and 107.9, similar to the carbon signal pattern of buanmycin (1), suggesting a polyaromatic system. Detailed one-dimensional (1D) and two-dimensional (2D) NMR spectroscopic analysis, including 1 H−1H COSY, HSQC, and HMBC measurements, was used to determine the structure of 2 (Figure 2). The HMBC correlations of the two methine protons (H-14, H-12) and two phenolic hydroxy protons (OH-9, OH-7) supported the elucidation of the anthraquinone moiety. The methine proton H-12 (δH 7.17) exhibited HMBC correlations to C-13 (δC 181.7), C-11 (δC 165.6), C-8a (δC 109.3), and C-10 (δC 107.9) and correlated with H-10 (δH 6.62) through meta coupling. HMBC correlations were observed from OH-9 (δH 12.15) to C-10 (δC 107.9) and C-8a (δC 109.3). Long-range heteronuclear coupling from OH-7 (δH 12.55) to C-7 (δC 158.0), C7a (δC 113.0), and C-6a (δC 132.3) was used to locate the hydroxy group at C-7. The downfield proton of H-14 (δH 8.83), which correlated with C-13 (δC 181.7), C-7a (δC 113.0), and C-6a (δC 132.3), established the anthraquinone moiety as one that incorporates two ketones at C-8 (δC 189.8) and C-13. The methylene signal protons H2-6 (δH 2.83) exhibited HMBC correlations to C-7 (δC 158.0) and C-6a (δC 132.3), connecting
Figure 1. Observed 13C−13C COSY correlations elucidating the carbon backbone of buanmycin (1).
see also the Supporting Information, Figure S6). Further analysis of the 1H−1H COSY and HMBC correlations was used to refine the backbone structure of 1 (Figure 2). The analysis of
Figure 2. Key 1H−1H COSY and HMBC correlations of 1 and 2.
the COSY NMR spectrum helped define the connectivity between the two methylenes (C-14 and C-15) and the orthocoupled aromatic methines (C-11 and C-12). The α-methyl serine unit was confirmed based on the HMBC correlations from the protons of the C-2a methyl group [H3-2a (δH 1.44)] to C-2 (δC 60.3), C-2b (δC 65.5), and C-1 (δC 174.2)]. This unit was established further by the HMBC coupling from H-2b to C-2, C-2a, and C-1. The long-range 1H−13C NMR correlations from H3-21 (δH 2.14) to the C-20 carbonyl (δC 205.8) and C-19 (δC 46.7) and from H2-19 to C-20 and C-18 (δC 133.1) confirmed the location of the C3 side chain at the left-end aromatic ring. The two-bond and three-bond HMBC correlations originating from the phenolic hydroxy groups (OH-7 and OH-9) determined their locations at C-7 and C-9, respectively. The methoxy signal was assigned to C-10 by the HMBC cross-peak between OCH3-10 (δH 3.87) and C-10 (δC 142.7). The heteronuclear correlations from H-11 and H-12 to C-9 and C-10, respectively, completed the right-end aromatic C
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Table 2. 1H and 13C NMR Data for Buanquinone (2) in DMSO-d6a position 1 2 3 4 4a 5 6 6a 7 7a 8 8a 9 10 11 12 12a 13 13a 14 14a 14b 15 16 17 OH-1 OH-7 OH-9 OH-11 a1
δC, type 155.9, 116.4, 137.3, 120.5, 140.3, 28.1, 20.5, 132.3, 158.0, 113.0, 189.8, 109.3, 164.5, 107.9, 165.6, 108.8, 135.5, 181.7, 130.2, 119.9, 141.0, 118.4, 49.6, 205.8, 29.7,
C CH C CH C CH2 CH2 C C C C C C CH C CH C C C CH C C CH2 C CH3
Table 3. Cytotoxicity Data IC50 for 1 and 2 against Selected Cancer Cell Lines
δH, mult. (J in Hz)
carcinoma
A549
HCT116
SNU638
K562
SKHEP1
MDA-MB231
1 2 etoposide
1.7 − 0.8
0.9 − 1.9
0.8 − 0.5
−a 18.3 3.1
1.9 − 1.1
1.2 − 10.6
6.73, s 6.65, s
a
2.73, m 2.83, m
IC50 > 100 μM.
the K562 cells. Buanquinone (2) exhibited moderate cytotoxicity against the K562 cell line but did not display any significant cytotoxicity against the other cell lines tested (Table 3). The antibacterial activities of the citreamicins against Grampositive bacteria have been reported previously. Therefore, the antibacterial activities of 1 and 2 were evaluated against Grampositive bacteria, including Staphylococcus aureus, Bacillus subtilis, and Kocuria rhizophila, as well as the Gram-negative pathogens Salmonella enterica and Proteus hauseri (Table 4).
6.62, d (2.0) 7.17, d (2.0)
Table 4. Antibacterial Activity Data for 1 and 2 MIC (μM) Gram-positive
8.83, s
3.71, s 2.16 10.39, 12.55, 12.15, 11.49,
a
s s s br s
Gram-negative
compound
S. aureus
B. subtilis
K. rhizophila
S. enterica
P. hauseri
1 2 ampicillin
10.5 −a 4.5
0.7 − 1.1
10.5 − 0.6
0.7 − 4.5
21.1 − 100 μg/mL.
The activity profile of 1 revealed that buanmycin is notably potent against the Gram-negative bacterium S. enterica, which causes salmonellosis. Buanquinone (2) did not demonstrate significant antibacterial activity. Additionally, buanmycin (1) exhibited moderate antifungal activity against C. albicans [minimum inhibitory concentration (MIC) = 21.1 μM]. In the antifungal assay against filamentous fungi, only buanmycin (1) displayed weak antifungal activity against A. f umigatus, with a MIC value of 84.3 μM. However, buanquinone did not exhibit significant antifungal activity. Since buanmycin (1) inhibited the growth of pathogenic bacteria, its biological activity was evaluated further in a Staphylococcus aureus sortase A inhibition assay. Sortase A is an enzyme from the transpeptidase family that plays a key role in adhesion and host invasion by Gram-positive bacteria.21 Notably, buanmycin inhibited Staphylococcus aureus sortase A and, with an IC50 value of 43.2 μM, was more potent than the positive control compound, p-hydroxymercuribenzoic acid (pHMB) (IC50: 104.4 μM).
H and 13C data were recorded at 600 and 125 MHz, respectively.
C-6 (δC 20.5) to C-6a. The observation of the COSY correlation between H2-6 and H-5 (δH 2.73) allowed the assignment of C-5 (δC 28.1) next to C-6. The HMBC correlations from H-5 to C-14b (δC 118.4), C-4a (δC 140.3), and C-4 (δC 120.5), from H-4 (δH 6.65) to C-4a (δC 140.3), and from OH-1 (δH 10.39) to C-14b (δC 118.4) indicated the entire cyclic system. In turn, the aliphatic chains that were linked to C-3 (δC 137.3) were indicated to have one methylene (δC 49.6-δH 3.71), one methyl group (δC 29.7-δH 2.16), and a carbonyl group (δC 205.8) by their HMBC correlations. Thus, the full structure of buanquinone (2) was completely assigned as a new pentacyclic anthraquinone. Even though buanmycin (1) shares a common xanthone feature with the citreamicins, including citreamicin ε A−B18 and citreamicin θ A−B,19 the pentacyclic (6/6/6/6/6) structure of 1 differs from the septacyclic (5/6/6/6/6/6/6) system in every citreamicin class of antibiotics. Buanquinone (2) is structurally related to KS-191-1, which originates from Streptomyces californicus,20 but lacks one carboxylic acid unit at C-2. The biological activities of 1 and 2 were evaluated in several bioassays. The cytotoxicities of 1 and 2 were tested against the following human carcinoma cell lines: A549 (lung cancer), HCT116 (colon cancer), SNU638 (gastric cancer), K562 (leukemia), SK-HEP1 (liver cancer), and MDA-MB231 (breast cancer) (Table 3). Buanmycin (1) exhibited potent cytotoxicity against the A549, HCT116, SNU638, SK-HEP1, and MDAMB-231 cells, with IC50 values of 0.8 to 1.9 μM, but not against
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EXPERIMENTAL SECTION
General Experimental Procedures. The optical rotations were measured with a JASCO P-200 polarimeter. The UV spectra were recorded on a PerkinElmer Lambda 35 UV/vis spectrometer. The IR spectra were acquired with a Thermo Nicolet iS10 spectrometer. The 1 H, 13C, and 2D NMR spectra were recorded on Bruker Avance 600 MHz spectrometers at the National Center for Interuniversity Research Facilities at Seoul National University (NCIRF) and at the Gachon University. The low-resolution electrospray ionization source mass spectra were obtained with an Agilent Technologies 6130 quadrupole mass spectrometer coupled to an Agilent Technologies 1200 series high-performance liquid chromatography (HPLC) instrument. High-resolution fast-atom bombardment (HRFAB) mass spectra were obtained using a JEOL JMS-600W high-resolution D
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optics. A total of 14 404 reflections, collected in the θ range 2.48° to 68.27°, yielded 8792 unique reflections (Rint = 0.0175), final R indices [I > 2σ(I)] R1 = 0.0327, wR2 = 0.0882. The structure was solved by direct methods (SHELXS-97) and refined by full-matrix least-squares on F2 (goodness of fit on F2 = 1. 060). Carbon, oxygen, and nitrogen atoms were refined anisotropically. Hydrogen atoms were placed in idealized locations except for two hydroxyl H atoms, which were refined freely with isotropic displacement parameters. The absolute stereochemistry was unambiguously determined as shown by the value of the refined Flack parameter. The asymmetric unit consists of two crystallographically independent, but chemically similar molecules. Absorption correction was performed by SADABS using with multiscan parameters. Crystallographic data for 1 have been deposited at the Cambridge Crystallographic Data Centre (CCDC 1018479). Copies of these data can be obtained free of charge via the Internet at www.ccdc.cam.ac.uk/conts/retrieving.html. Crystal data of buanmycin (1): C34H35NO14, M = 681.63, triclinic crystal system, crystal size 0.27 × 0.21 × 0.08 mm3, space group P1, a = 7.4957(3) Å, b = 12.0298(4) Å, c = 17.8347(7) Å, α = 87.3870(12)°, β = 87.5550(11)°, γ = 74.7080(10)°, V = 1548.85(10) Å3, Z = 2, μ = 0.970 mm−1, Dc = 1.462 g/cm3. F(000) = 716, Flack parameter x = −0.02(12). Cytotoxicity Assay. The cytotoxicity for cultured cancer cells of compounds 1 and 2 was evaluated with a sulforhodamine B (SRB) assay. Six human cancer cell lines (HCT116, MDA-MB231, SNU638, A549, K562, and SK-HEP1) were seeded into 96-well plates at 3 × 104 cells/mL and incubated with various concentrations of each compound at 37 °C in a humidified atmosphere with 5% CO2. After 72 h, the cells were fixed with a 10% TCA solution for 1 h, and the cellular proteins were stained with 0.4% SRB in 1% acetic acid. The stained cells were dissolved in 10 mM Tris buffer (pH 10.0). The effects of 1 and 2 on cell cytotoxicity were calculated as a percentage relative to the solvent-treated control. The IC50 values were determined by nonlinear regression analysis (percent survival versus concentration). Etoposide was used as the positive control. Antibacterial Activity Assay. Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, and Kocuria rhizophila NBRC 12708) and Gram-negative bacteria (Salmonella enterica ATCC 14028, Proteus hauseri NBRC 3851, and Escherichia coli ATCC 25922) were used for the antimicrobial activity tests.22 The bacteria were incubated overnight in Luria−Bertani (LB) broth at 37 °C, harvested by centrifugation, and washed twice with sterile distilled water. The compound solutions were prepared in DMSO. Each solution was diluted with Plate Count Broth (Difco) to generate serial 2-fold dilutions in the range of 50 to 0.8 μg/mL. Aliquots (10 μL) of the broth containing the test bacteria were added to each 96-well microtiter plate at a final concentration of 5 × 105 colony-forming units (cfu)/mL. The plates were incubated for 12 h at 37 °C. The MIC values were determined as the lowest concentration at which there is no visible growth. Ampicillin was used as a reference compound. Antifungal Activity Assay. Candida albicans ATCC 10231 was cultivated with YPD medium (1% yeast extract, 2% peptone, and 2% dextrose). After incubation for 48 h at 28 °C, the cells were harvested by centrifugation and washed twice with sterile distilled water. Aspergillus f umigatus HIC 6094, Trichophyton rubrum NBRC 9185, and T. mentagrophytes IFM 40996 were plated onto potato dextrose agar plates and incubated for 2 weeks at 28 °C. Spores were harvested and washed twice with sterile water, and then the spores were resuspended in water to an initial inoculum concentration of 5 × 105 spores/mL. In each well of a 96-well plate, 10 μL of the cell suspension was mixed with 90 μL of potato dextrose broth (Difco) (5 × 104 cells/mL) containing the test compound in DMSO solutions (final concentration, 0.5%).22 A culture containing DMSO (0.5%) and a culture supplemented with amphotericin B were used as a solvent control and as a positive control, respectively. Sortase A Inhibition Assay. Staphylococcus aureus ATCC 6538p was the source of the srtA gene. The expression and purification of recombinant SrtAΔ24 were carried out according to a previously published procedure.23 The reactions were performed in 100 μL
mass spectrometer at the NCIRF. The semipreparative HPLC separations were performed with a Gilson 305 pump and a Gilson UV/vis-155 detector. Bacterial Isolation and Cultivation. The sample was collected in June 2011 from a tidal mud flat in Buan, Republic of Korea. Dried sediment was pressed onto the surface of actinomycete isolation medium (1 L of seawater, 18 g of agar, 100 mg/L cycloheximide) and A5 (750 mL of seawater, 250 mL of distilled water, 18 g of agar, 100 mg/L cycloheximide), A6 (1 L of seawater, 18 g of agar, and 5 mg/L polymyxin B sulfate), and A7 (1 L of seawater, 18 g of agar, and 5 mg/ L kanamycin) media agar plates using an autoclaved foam plug (2 cm in diameter). Then, 50 μL of the suspension, the mixture of 0.5 g of dried sediment and 10 mL of sterilized artificial seawater, was heated to 55 °C and inoculated onto the agar plates. The plates were incubated at 25 °C for 3 weeks. The strain SNR69 was isolated from the actinomycete isolation medium plates. The sequence analysis of the 16S rDNA sequencing analysis data (GenBank accession number: KM353585) obtained from COSMO Co., Ltd. revealed that strain SNR69 was most similar to Streptomyces cyaneus (99% identity). SNR69 was cultivated in 1 L of YEME medium (4 g of yeast extract, 10 g of malt extract, 4 g of glucose, and 27 g of artificial sea salt) at 170 rpm and 30 °C for 5 days. Altogether 20 L of bacterial liquid culture was prepared. Extraction and Isolation. The entire culture was extracted twice with 30 L of ethyl acetate. The EtOAc extract was concentrated in vacuo to yield 7 g of dry material. One-half of the dried extract was fractionated with 150 mL each of 20%, 40%, 60%, 80%, and 100% MeOH in water using a packed C18 (20 g) column. After the fractionation, buanmycin (1) was identified in the 60% and 80% fractions, and buanquinone (2) was detected in the 80% and 100% fractions. Each fraction was then subjected to reversed-phase HPLC (Kromasil 100-5-C18 250 × 10 mm, flow rate 2 mL/min, UV 280 nm detection, with a 50−60% aqueous acetonitrile gradient, 0.1% formic acid, over 60 min). Under these purification conditions, buanmycin (1) and buanquinone (2) eluted at 10 and 51 min, respectively. Finally, the procedures were repeated to afford pure buanmycin (1) (22 mg) and buanquinone (2) (6 mg). Buanmycin (1): yellow powder; [α]25D +72 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 216 (2.35), 239 (2.21), 276 (2.33), 297 (2.64), 424 (0.28) nm; IR (neat) νmax 3361, 2851, 1706, 1630, 1491, 1282, 1038 cm−1; 1H and 13C NMR data, see Table 1; HRFABMS m/z 594.1613 [M + H]+ (calcd for C30H28NO12, 594.1612). Buanquinone (2): red powder; [α]25D +128 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 206 (1.20), 309 (0.89), 473 (0.40) nm; IR (neat) νmax 3841, 3394, 2928, 1681, 1442, 1208, 1140 cm−1; 1H and 13 C NMR data, see Table 2; HRFABMS m/z 431.1139 [M + H]+ (calcd for C25H19O7, 431.1131). 13 C Labeling of Buanmycin (1). The bacterial strain SNR69 was cultivated in 50 mL of YEME medium. After 3 days on a rotary shaker at 170 rpm and 30 °C, 10 mL of the culture was inoculated into 200 mL of a 13C-enriched medium (0.8 g of uniformly labeled 13C-glucose, 0.8 g of yeast extract, 2 g of malt extract, and 5.4 g of artificial sea salt). In total, 3.2 L (16 each × 200 mL) was cultivated for 5 days. The 13Cenriched buanmycin was purified as described above. 13 C−13C COSY NMR Spectroscopy. The 13C−13C COSY NMR experiment was performed in a constant-time fashion to remove the vicinal carbon−carbon splitting in the t1 dimension. The 180° inversion of the carbon nuclei was achieved using an adiabatic pulse for broadband applications (34722 Hz). The proton spins were decoupled using a WALTZ-65 supercycle during the entire pulse sequence, except for a recycle delay of 2 s. The total number of points was 1024 (t2) × 256 (t1) complex points, and the spectral widths for both dimensions were 230 ppm. The entire experiment required 2 days and 8 h with 9 mg of buanmycin (1). Crystallization and X-ray Crystallographic Analysis of Buanmycin (1). Buanmycin was crystallized by slow evaporation of a solution in EtOAc−hexane. The crystal structure and absolute configuration of 1 were determined using data collected at T = 100(2) K with Cu Kα radiation (λ = 1.54178 Å) on a Bruker D8 diffractometer with an FR-591 rotating-anode source and microfocus E
dx.doi.org/10.1021/np500736b | J. Nat. Prod. XXXX, XXX, XXX−XXX
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containing 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, 5 mM CaCl2, 1 μg of fluorescent peptide (H2N-Abz-LPXTG-Dap(Dnp)-NH2), 1 mM pentaglycine, 24 μg of recombinant SrtAΔ24, and the test compounds at various concentrations (0.5% DMSO final concentration). Appropriate blanks contained all of the above constituents, with the exception of the test sample. The reactions were performed for 1 h at 37 °C. The sample fluorescence was measured using emission and excitation wavelengths of 420 and 317 nm, respectively. pHydroxymercuribenzoic acid, a known sortase inhibitor,24 was used as a positive control.
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(16) (a) Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem. 1997, 69, 3346−3352. (b) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem. 1997, 69, 5146−5151. (17) Miller, E. D.; Kauffman, C. A.; Jensen, P. R.; Fenical, W. J. Org. Chem. 2007, 72, 323−330. (18) (a) Carter, G. T.; Nietsche, J. A.; Williams, D. R.; Borders, D. B. J. Antibiot. 1990, 43, 504−512. (b) Liu, L.-L.; He, L.-S.; Xu, Y.; Han, Z.; Li, Y.-X.; Zhong, J.-L.; Guo, X.-R.; Zhang, X.-X.; Ko, K. M.; Qian, P.-Y. Chem. Res. Toxicol. 2013, 26, 1055−1063. (19) Liu, L.-L.; Xu, Y.; Han, Z.; Li, Y.-X.; Lu, L.; Lai, P.-Y.; Zhong, J.L.; Guo, X.-R.; Zhang, X.-X.; Qian, P.-Y. Mar. Drugs 2012, 10, 2571− 2583. (20) (a) Matsuda, Y.; Kase, H. J. Antibiot. 1987, 40, 1104−1110. (b) Yasuzawa, T.; Yoshida, M.; Shirahata, K.; Sano, H. J. Antibiot. 1987, 40, 1111−1114. (21) Hendrickx, A. P.; Budzik, J. M.; Oh, S.-Y.; Schneewind, O. Nat. Rev. Microbiol. 2011, 9, 166−176. (22) Oh, K.-B.; Lee, J. H.; Chung, S.-C.; Shin, J.; Shin, H. J.; Kim, H.K.; Lee, H.-S. Bioorg. Med. Chem. Lett. 2008, 18, 104−108. (23) Oh, K.-B.; Kim, S.-H.; Lee, J.; Cho, W.-J.; Lee, T.; Kim, S. J. Med. Chem. 2004, 47, 2418−2421. (24) Ton-That, H.; Liu, G.; Mazmanian, S. K.; Schneewind, O. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12424−12429.
ASSOCIATED CONTENT
S Supporting Information *
The detailed experimental procedures and NMR data for compounds 1 and 2 and X-ray crystallographic data of 1 are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*(D.-C. Oh) Tel: +82-2-880-2491. Fax: +82-2-762-8322. Email: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Prof. C. C. Hughes, University of California, San Diego, for assistance with the X-ray crystallographic analysis. This work was supported by National Research Foundation of Korea grants funded by the Korean Government (Ministry of Science, ICT and Future Planning) (M1A5A1-2010-0020429 and 2009-0083533).
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DEDICATION Dedicated to Dr. William Fenical of Scripps Institution of Oceanography, University of California−San Diego, for his pioneering work on bioactive natural products.
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