Note pubs.acs.org/jnp
Bacterial Production of a Pederin Analogue by a Free-Living Marine Alphaproteobacterium Carmen Schleissner, Librada M. Cañedo, Pilar Rodríguez, Cristina Crespo, Paz Zúñiga, Ana Peñalver, Fernando de la Calle,* and Carmen Cuevas Research and Development Department, PharmaMar S.A., Avenida de los Reyes, 1, Colmenar Viejo 28770, Madrid, Spain S Supporting Information *
ABSTRACT: The polyketide pederin family are cytotoxic compounds isolated from insects, lichen, and marine sponges. During the past decade, different uncultivable bacteria symbionts have been proposed as the real producers of these compounds, such as those found in insects, lichen, and marine sponges, and their trans-AT polyketide synthase gene clusters have been identified. Herein we report the isolation and biological activities of a new analogue of the pederin family, compound 1, from the culture of a marine heterotrophic alphaproteobacterium, Labrenzia sp. PHM005. This is the first report of the production of a pederin-type compound by a free-living marine bacteria that could be cultured in the laboratory.
P
other pederin-type compounds, psymberin has shown selective activity against numerous tumor types.13 The biosynthetic connection between these structurally related families of bioactive toxins has been widely discussed by Piel and co-workers, who have demonstrated the existence of putative trans-AT polyketide synthase gene clusters for pederins, onnamides, psymberin, diaphorin, and nosperin in the genomes of different bacterial symbionts.14 However, this fascinating story of the broad distribution of symbionts does not finish here. During our research to explore cultivable bacterial biodiversity isolated from marine samples, we have isolated compound 1, representing a new pederin structure, from the cultures of a free-living and strictly marine bacterium, the alphaproteobacterium Labrenzia sp. PHM005, isolated from a marine sediment. In other recent examples, phylogenetically related marine alphaproteobacteria have been reported as the real producers of bioactive metabolites initially assigned to be produced by marine tunicates. Such is the case of the free-living Tistrella mobilis strains, isolated from different marine sources by our own research group15 as well as two other research groups,16 which are able to produce the didemnins, marine peptides isolated several decades ago from Caribbean tunicates.17 This is the first ever report of the production of a pederintype compound by a free-living wild-type bacterium that can be cultured in the laboratory. The production of 1 can be modulated by changes to the fermentation conditions. Labrenzia sp. PHM005 was isolated on sea salt agar medium from a marine sediment sample collected in the Indian Ocean. Observation of the cells by transmission electron microscopy
ederin (2) is the best known cytotoxin belonging to a large family of potent tetrahydropyran core polyketides.1 This compound and similar analogues, such as pseudopederin (3) and pederone (4) as minor components,3 have all been isolated from rove beetles.2 Recently a new pederin-type compound, diaphorin (5), has been isolated from the psyllid insect Diaphorina citri.4
Surprisingly, similar bioactive tetrahydropyran structures have also been isolated from diverse marine sponges such as in the case of mycalamides A and B in Mycale,5 mycalamides C and D in Stylinos,6 the large family of theopederins A−J in Theonella,7 theopederins K and L in Discodermia,8 and onnamides in Theonella9 and Trachycladus.10 However, the most widely studied pederin-type family is the psymberins or orirciniastatins, isolated from the marine sponges Psammocinia11 and Ircinia.12 In contrast to the equipotent toxicity of the © 2017 American Chemical Society and American Society of Pharmacognosy
Received: May 11, 2017 Published: July 11, 2017 2170
DOI: 10.1021/acs.jnatprod.7b00408 J. Nat. Prod. 2017, 80, 2170−2173
Journal of Natural Products
Note
allowed us to identify motile rods (0.6−0.8 μm wide and 1.6− 2.1 μm long) with a single, subpolar inserted flagellum (Figure 1).
second-order polynomial regression confirmed that maximum activity could be achieved using a combination of mannitol (76 g/L), brewer’s yeast Sentient G20 (17.6 g/L), CaCO3 (13 g/ L), and marine salts (36 g/L) in addition to other minor elements as described for medium 16B/d in the Experimental Section. This medium was selected for further scale-up and an interval of between 96 and 120 h defined as the proper time course for extraction of the supernatant. Once the optimum fermentation conditions were defined, a 50 L scale-up culture was centrifuged to separate cells and the supernatant. The supernatant was extracted at pH 7.2 with EtOAc, and after freezing the mixture overnight at −20 °C the organic phase was filtered to remove ice and dried to give 1.8 g of a brown extract. The extract was further purified by silica gel vacuum flash chromatography (VFC) and semipreparative reversed-phase HPLC, yielding 24.5 mg of 1. The structure of 1 was elucidated using 1D NMR (1H and 13 C), 2D NMR (COSY, TOCSY, gHSQC, gHMBC, and ROESY), MS analysis, and specific rotation data. Compound 1 was obtained as a colorless oil, and its molecular formula established as C24H43NO9 by (+)-HRES-TOFMS in combination with 1H and 13C NMR spectroscopic data. The 1H NMR spectrum in CDCl3 revealed signals of a NH amide group at δH 7.18, exocyclic methylene protons (δH 4.85 and 4.73), three methoxy groups (δH 3.32, 3.38, and 3.32), seven oxymethine protons (δH 5.37, 4.32, 3.99, 3.83, 3.64, 3.36, and 3.31), one oxymethylene group (δH 3.65 and 3.48), and four methyl groups (δH 1.19, 1.01, 0.95, and 0.88). Additionally, 13C NMR data of 1 exhibited one carbonyl carbon signal at δC 171.9, one ketal carbon signal at δC 99.7, and one exocyclic double bond at δC 145.7 (C-4) and δC 110.5 (C-21). Analysis of the COSY and TOCSY spectra showed correlations from H3-19 to H3-20, H10 to H-13, and H-15 to H2-18 and correlations from the NH signal at δH 7.18 to H-10. Analysis of the HMBC data confirmed that the three methoxy groups at δH 3.32, 3.38, and 3.32 are attached to C-6, C-10, and C-17, respectively. After detailed analysis of 2D NMR data, COSY, TOCSY, and HMBC, the remainder of the carbon skeleton was structurally related to those of the pederin class of compounds,6,8,21,22 with the structure of 1 being very similar to pederin,8,21 except for the absence of the O-methyl group at the C-18 position. As such, we have identified 1 as 18-O-demethylpederin. The relative configuration of 1 was confirmed on the basis of ROESY data and analysis of coupling constants. The specific rotation of 1 ([α]20D +82.4, c 0.49, CHCl3) showed the same sign as pederin ([α]20D +86.8, c 1.00, CHCl3).22 The absolute configuration of pederin has been established by an X-ray crystallographic study6 and stereoselective synthesis.22 On this basis, we tentatively propose the absolute configuration of 1 to be the same as pederin and other reported analogue compounds.23 The antiproliferative activity of 1 was assessed in a panel of four human tumor cell lines using the sulforhodamine B (SRB)24 methodology adapted to provide a quantitative measurement of the cell growth and viability. The GI50 (compound concentration that produces 50% cell growth inhibition, as compared to control cultures) obtained for 1 for the four cell lines was calculated to be in the range (2.0−2.9) × 10−09 M. As reported for the rest of pederin-type families, except for psymberin,13 there was no cell cytotoxic selectivity.
Figure 1. Electron microscopy (negative staining) of Labrenzia sp. PHM005. Bar: 0.2 μm. Arrow: Detached flagellum.
The bacterium is clearly marine salt dependent, as it needs more than 2.5% NaCl to grow, with the optimal concentration of marine salt for production of 1 being 36 g/L, similar to ocean conditions. Colonies on Marine Agar 2216 (Difco) are beige, almost transparent, and smooth with an entire margin. After 3 weeks the colonies become darker brown, possibly due to the effect of bacteriochlorophyll a and carotenoid production, as described for Labrenzia alexandrii DFL-11T, the closest type strain,18 showing 98.6% similarity based on analysis of the 1355 bp 16S rRNA against the SILVA LTPs123 database.19 Furthermore, its systematic relationship with L. alexandrii was confirmed based on a neighbor-joining analysis using the 16S rRNA gene sequence. Sequences from the sister species were obtained from the SILVA LTPs123 (Figure 2).
Figure 2. Neighbor-joining tree based on 16S rRNA gene sequences that show the relationship between PHM005 and the type strains of closely related species of the genera Labrenzia and Stappia. Bootstrap value >99%.
The effect of various media components and time course of the culture of Labrenzia sp. PHM005 was examined using the cytotoxicity effect as the measure of production. The first-order model based on the Placket−Burman20 design showed that mannitol, entire inactive yeasts, calcium carbonate, marine salts, inoculum development, temperature, and the time of sampling significantly influenced the antiproliferative potency. The 2171
DOI: 10.1021/acs.jnatprod.7b00408 J. Nat. Prod. 2017, 80, 2170−2173
Journal of Natural Products
■
Note
compound 1 with a retention time of 25.0 min at these HPLC conditions. 18-O-demethylpederin (1): colorless oil; [α]20D +82.4 (c 0.49, CHCl3) and [α]20D +81.3 (c 0.36, MeOH); 1H NMR (CDCl3) δH 3.99 (1H, dq, J = 6.6, 2.7 Hz, H-2), 2.25 (1H, d, J = 7.1, 2.7 Hz, H-3), 2.43 (1H, d, J = 14.1 Hz, H-5a), 2.36 (1H, dt, J = 14.1, 2.7 Hz, H-5b), 4.31 (1H, s, H-7), 7.18 (1H, d, J = 9.8 Hz, NH), 5.37 (1H, dd, J = 9.8, 7.8 Hz, H-10), 3.83 (1H, dt, J = 7.8, 2.7 Hz, H-11), 2.04 (1H, dt, J = 13.5, 3.6 Hz, H-12a), 1.75 (1H, m, H-12b), 3.64 (1H, m, H-13), 3.31 (1H, m, H-15), 1.75 (1H, m, H-16a), 1.57 (1H, dd, J = 14.3, 9.7 Hz, H16b), 3.36 (1H, m, H-17), 3.65 (1H, m, H-18), 3.48 (1H, m, H-18), 1.19 (3H, d, J = 6.6 Hz, H-19), 1.01 (3H, d, J = 7.1 Hz, H-20), 4.85 (1H, s, H-21), 4.73 (1H, s, H-21), 0.95 (3H, s, C-22), 0.88 (3H, s, C23), 3.32 (3H, s, MeO-6), 3.38 (3H, s, MeO-10), 3.32 (3H, s, MeO17); 13C NMR (CDCl3) δC 69.6 (d, C-2), 41.3 (d, C-3), 145.7 (s, C4), 34.1 (t, C-5), 99.7 (s, C-6), 73.1 (d, C-7), 171.9 (s, C-8), 79.4 (d, C-10), 72.6 (d, C-11), 29.6 (t, C-12), 71.8 (d, C-13), 38.4 (s, C-14), 75.4 (d, C-15), 29.2 (t, C-16), 79.0 (d, C-17), 63.8 (t, C-18), 17.9 (q, C-19), 12.0 (q, C-20), 110.5 (t, C-21), 23.1 (s, C-22), 13.5 (s, C-23), 49.1 (q, MeO-6), 56.4 (q, MeO-10), 56.6 (q, MeO-17); (+)-ESIMS m/z 512.3 [M + Na]+; (+)-HRES-TOFMS m/z 512.2873 [M + Na]+ (calcd for C24H43NO9Na, 512.2830). Cytotoxic Activity. For the antiproliferative experiments described for 1, four human tumor cell lines were used: A549 (ATCC CCL-185) (lung carcinoma, NSCLC); HT-29 (ATCC HTB-38) (colon adenocarcinoma); MDA-MB-231 (ATCC HTB-26) (breast adenocarcinoma); and PSN-1 (ATCC CRL-3211) (pancreas adenocarcinoma). All the cell lines were obtained from the American Type Culture Collection (ATCC) and derive from different types of human cancer. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM Lglutamine, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C, 5% CO2, and 98% humidity. For the experiments, cells were harvested from subconfluent cultures using trypsinization and resuspended in fresh medium before counting and plating. Cells were seeded in 96-well microtiter plates, at 5000 cells per well in aliquots of 150 μL, and allowed to attach to the plate surface for 18 h (overnight) in drug-free medium. After that, one control (untreated) plate of each cell line was fixed (as described below) and used for time zero reference value. Culture plates were then treated with test compounds (50 μL aliquots of 4× stock solutions in complete culture medium plus 4% DMSO) using ten 2/5 serial dilutions (concentrations ranging from 10 to 0.003 μg/mL) and triplicate cultures (1% final concentration in DMSO). After 72 h of treatment, the effects on cell growth and survival were estimated by applying the NCI algorithm.27
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were determined using a Jasco P-1020 polarimeter. NMR spectra were obtained on a Varian “Unity 500” spectrometer at 500/125 MHz (1H/13C) and on a Varian “Unity 400” spectrometer at 400/100 MHz (1H/13C). Chemical shifts were reported in ppm using residual CDCl3 (δ 7.26 ppm for 1H and 77.16 ppm for 13C) as an internal reference. (+)ESIMS data were recorded using an Agilent 1100 Series LC/MSD spectrometer. High-resolution mass spectrometry (HRMS) was performed on an Agilent 6230 TOF LC/MS system using the ESIMS technique. Bacteria Isolation. The pederin-type producing bacterium Labrenzia sp. PHM005 was isolated from a marine sediment collected at a depth of 18 m on the coast of Kenya in 2005. Approximately 5 g of sea gravel material was collected in a 50 mL Falcon tube containing sterile artificial seawater (ASW) and was maintained at 5 °C for 5 days before being processed. Once in the laboratory, the sample was homogenized, and 100 μL of a 1:100 dilution with ASW spread directly on Petri dishes with a sea salt medium consisting of 27 g/L marine salts (Tropic Marin PRO-REEF), 16 g/L agar, and 0.2 mg/mL cycloheximide. After incubation for 3 weeks at 28 °C, a slightly brown colony was picked and transferred to the same sea salt medium to confirm the purity and generate biomass for molecular characterization, with one colony being inoculated on liquid marine broth for further conservation on 20% glycerol at −80 °C as a cell bank. Electron Microscopy. Cells in the midexponential growth phase were adsorbed on 400-mesh carbon-collodion-coated grids for 2 min, negatively stained with 2% uranyl acetate, imaged with a Jeol JEM 1011 transmission electron microscope operated at 100 kV, and photographed with a CCD Gatan Erlangshen ES1000W camera. 16S rRNA Characterization. For DNA extraction the strain was grown in marine broth (DIFCO 1196) for 72 h. Cells were recovered and lysed by boiling with 4% NP40 for 10 min. Cell debris was discarded by centrifugation. The 16S rDNA gene was amplified by the polymerase chain reaction using the bacterial primers F1 and R5.25 The amplified nearly full-length 16S rRNA gene sequence of 1355 bp was submitted to GenBank with accession number SUB1740132. Evolutionary analyses were carried out including the 16S gene from different taxa (SILVA LTPs123 database).19 A neigbor-joining analysis was performed using 1000 replicates in MEGA7.26 Only the results with a bootstrap of >99% were indicated in the tree. Cultivation and Extraction. After culture, the whole broths were lyophilized and extracted with MeOH/acetone/H2O (1:1:0.2). A 0.5 mL sample of the crude extract was dried and screened for cytotoxic activity. The best cytotoxic activity was achieved in the 16B/d medium at 120 h. This medium consisted of 17.5 g/L of brewer’s yeast (Sensient, G2025), 76 g/L mannitol, 7 g/L (NH4)2SO4, 13 g/L CaCO3, 0.09 g/L FeCl3, and 36 g/L marine salts (Tropic Marin PROREEF). A 50 L scale-up of this bacterium in 16B/d medium was prepared in 200 × 2 L Erlenmeyer flasks each with a working volume of 250 mL. The production flasks were inoculated with 2% of the bacteria grown for 72 h in marine broth (Difco 1196) from another highly grown preinoculum. The scale-up was incubated for 120 h at 28 °C in a rotatory shaker at 220 rpm with 5 cm eccentricity. The culture was then centrifuged at 8000 rpm for 20 min to give 45 L of aqueous supernatant, which was extracted twice with EtOAc, and the organic phase was dried to give an organic extract. Isolation of 1. The extract was applied to a silica gel VFC system, using a stepwise gradient elution with n-hexane/EtOAc and EtOAc/ MeOH mixtures to give 11 fractions. The active fractions (550 mg) were eluted with EtOAc and EtOAc/MeOH (9:1) and were subjected to preparative reversed-phase HPLC equipped with a Symmetry C18 column (19 × 150 mm, 7 μm) using a linear gradient of H2O/CH3CN from 5% to 35% of CH3CN in 30 min at a flow rate of 13.5 mL/min, to afford a very active peak-fraction (77 mg) with a retention time of 24.5 min containing 1 based on the HPLC-MS chromatogram. This fraction was further purified by semipreparative HPLC on an XBridge C18 column (10 × 250 mm, 5 μm) using an isocratic elution with H2O/CH3CN (78:22) at a flow of 4 mL/min, to yield 24.5 mg of pure
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00408. 1 H NMR, 13C NMR, gHSQC, gCOSY, TOCSY, gHSQC, gHMBC, LR-HSQMBC, and ROESY spectra of compound 1 (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel: +34 91 846 6027. Fax: +34 91 846 6001. E-mail:
[email protected]. ORCID
Librada M. Cañedo: 0000-0002-1273-4138 Fernando de la Calle: 0000-0001-8161-8299 Notes
The authors declare no competing financial interest. 2172
DOI: 10.1021/acs.jnatprod.7b00408 J. Nat. Prod. 2017, 80, 2170−2173
Journal of Natural Products
■
Note
(16) (a) Tuskimoto, M.; Nagaoka, M.; Shishido, Y.; Fujimoto, J.; Nishisaka, F.; Matsumoto, S.; Harunari, E.; Imada, C.; Matsuzaki, T. J. Nat. Prod. 2011, 74, 2329−2331. (b) Xu, Y.; Kersten, R. D.; Nam, S.J.; Lu, L.; Al-Suwailem, A. M.; Zheng, H.; Fenical, W.; Dorrestein, P. C.; Moore, B. S.; Qian, P.-Y. J. Am. Chem. Soc. 2012, 134, 8625−8632. (17) Rinehart, K. L.; Gloer, J. B.; Cook, J. C.; Mizsak, S. A.; Scahill, T. A. J. Am. Chem. Soc. 1981, 103, 1857−1859. (18) Biebl, H.; Pukall, R.; Lünsdorf, H.; Schulz, S.; Allgaier, M.; Tindall, B. J.; Wagner-Döbler, I. Int. J. Syst. Evol. Microbiol. 2007, 57, 1095−1107. (19) Yarza, P.; Richter, M.; Peplies, J.; Euzeby, J.; Amann, R.; Schleifer, K. H.; Ludwig, W.; Glöckner, F. O.; Rosselló-Mora, R. Syst. Appl. Microbiol. 2008, 31, 241−250. (20) Plackett, R. L.; Burman, J. P. Biometrika 1946, 33, 305−352. (21) (a) Furusaki, A.; Watanabe, T.; Matsumoto, T.; Yanagiya, M. Tetrahedron Lett. 1968, 9, 6297−6300. (b) Matsumoto, T.; Yanagiya, M.; Yasuda, S. Tetrahedron Lett. 1968, 9, 6301−6304. (22) Matsuda, F.; Tomiyoshi, N.; Yanagiya, M.; Matsumoto, T. Tetrahedron 1988, 44, 7063−7080. (23) Wan, S.; Wu, F.; Rech, J. C.; Green, M. E.; Balachandran, R.; Seth Horne, W.; Day, B.; Floreacing, P. E. J. Am. Chem. Soc. 2011, 133, 16668−16679. (24) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112. (25) Cook, A. E.; Myers, P. R. Int. J. Syst. Evol. Microbiol. 2003, 53, 1907−1915. (26) Kumar, S.; Stecher, G.; Tamura, K. Mol. Biol. Evol. 2016, 33, 1870−1874. (27) Shoemaker, R. H. Nat. Rev. Cancer 2006, 6, 813−823.
ACKNOWLEDGMENTS We gratefully acknowledge the help of our PhamaMar colleagues, C. de Eguilior for collecting the marine sediment, ́ for work with the cultures, J. Garciá and B. Delgado X. Benitez ́ for their technical assistance, J. M. Dominguez for design of the biological assays, and S. Munt for revision of the manuscript. The present research was financed in part by a Grant from the EU Horizon 2020 Project INMARE 634486 and by the Minister of Economy and Competitiveness of Spain under the program RETOS-COLABORACIÓ N with the project number RTC-2016-4892-1 (DESPOL).
■
REFERENCES
(1) Mosey, R. A.; Floreancig, P. E. Nat. Prod. Rep. 2012, 29, 980− 995. (2) Narquizian, R.; Kocienski, P. J. In The Role of Natural Products in Drug Discovery; Mulzer, R.; Bohlmann, R., Eds.; Springer-Verlag: Heidelberg, Germany, 2000; Vol. 32, pp 25−56. (3) Cardani, C.; Ghiringhelli, D.; Mondelli, R.; Quilico, A. Tetrahedron Lett. 1965, 29, 2537−2545. (4) Nakabachi, A.; Ueoka, R.; Oshima, K.; Teta, R.; Mangoni, A.; Gurgui, M.; Oldham, N. J.; van Echten-Deckert, G.; Okamura, K.; Yamamoto, K.; Inoue, H.; Ohkuma, M.; Hongoh, Y.; Miyagishima, S. Y.; Hattori, M.; Piel, J.; Fukatsu, T. Curr. Biol. 2013, 23, 1478−1484. (5) (a) Perry, N. B.; Blunt, J. W.; Munro, M. H. J. Am. Chem. Soc. 1988, 110, 4850−4851. (b) Perry, N. B.; Blunt, J. W.; Murray, M. H.; Thomson, A. M. J. Org. Chem. 1990, 55, 223−227. (6) Simpson, J. S.; Garson, M. J.; Blunt, J. W.; Munro, M. H. J. Nat. Prod. 2000, 63, 704−706. (7) (a) Fusetani, N.; Sugawara, T.; Matsunaga, S. J. Org. Chem. 1988, 57, 3828−3832. (b) Tsukamoto, S.; Matsunaga, S.; Fusetani, N.; Tohe, A. Tetrahedron 1999, 55, 13697−13702. (8) Paul, G. K.; Gunasekera, S. P.; Longley, R. E.; Pomponi, S. A. J. Nat. Prod. 2002, 65, 59−61. (9) (a) Sakemi, S.; Ichiba, T.; Kohmoto, S.; Saaucy, G.; Higa, T. J. Am. Chem. Soc. 1988, 110, 4851−4853. (b) Matsunaga, S.; Fusetani, N.; Nakao, Y. Tetrahedron 1992, 48, 8369−8376. (c) Kobayashi, J.; Itagaki, F.; Shigemori, H.; Sasaki, T. J. Nat. Prod. 1993, 56, 976−981. (10) Vuong, D.; Capon, J.; Lacey, E.; Gill, J. H.; Heiland, K.; Friedel, T. J. Nat. Prod. 2001, 64, 640−642. (11) Cichewicz, R. H.; Valeriote, F. A.; Crews, P. Org. Lett. 2004, 6, 1951−1954. (12) Pettit, G. R.; Xu, J. P.; Chapuis, J. C.; Pettit, R. K.; Tackett, L. P.; Doubek, D. L.; Hooper, J. A.; Schmidt, J. M. J. Med. Chem. 2004, 47, 1149−1152. (13) Wu, C. H.; Feng, Y.; Cardenas, E.; Williams, N.; Floreacing, P. E.; De Brabander, J. K.; Roth, M. G. J. Am. Chem. Soc. 2012, 134, 18998−19003. (14) (a) Piel, J.; Höfer, I.; Hui, D. J. Bacteriol. 2004, 186, 1280−1286. (b) Wilson, M. C.; Mori, T.; Rückert, C.; Uria, A.; Helf, M. J.; Takada, K.; Gernert, C.; Steffens, U. A.; Heycke, N.; Schmitt, S.; Helfrich, E. J.; Brachmann, A. O.; Gurgui, C.; Wakimoto, T.; Krachy, M.; Crüsemann, M.; Hentschel, U.; Abe, I.; Matsunaga, S.; Kalinowski, J.; Takeyama, H.; Piel, J. Nature 2014, 506, 58−62. (c) Fisch, K. M.; Gurgui, C.; Heycke, N.; van der Sar, S. A.; Anderson, S. A.; Webb, V. L.; Taudien, S.; Platzer, M.; Rubio, B. K.; Robinson, S. J.; Crews, P.; Piel, J. Nat. Chem. Biol. 2009, 5, 494−501. (d) Nakabachi, A.; Ueoka, R.; Oshima, K.; Teta, R.; Mangoni, A.; Gurgui, M.; Oldham, N. J.; van EchtenDeckert, G.; Okamura, K.; Yamamoto, K.; Inoue, H.; Ohkuma, M.; Hongoh, Y.; Miyagishima, S. Y.; Hattori, M.; Piel, J.; Fukatsu, T. Curr. Biol. 2013, 23, 1478−1484. (e) Kampa, A.; Gagunashvili, A. N.; Gulder, T. A.; Morinaka, B.; Daolio, C.; Godejohann, M.; Miao, V. P.; Piel, J.; Andrésson, O. S. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 3129−3137. (15) Schleissner, C.; Rodríguez, P.; Zuñiga, P.; Peñalver, A.; García, J.; Delgado, B.; de la Calle, F.; Cuevas, C. Poster. MaNaPro. 2013, La Toja, Galicia, Spain. 2173
DOI: 10.1021/acs.jnatprod.7b00408 J. Nat. Prod. 2017, 80, 2170−2173
