Note pubs.acs.org/jnp
Dermacozines H−J Isolated from a Deep-Sea Strain of Dermacoccus abyssi from Mariana Trench Sediments Marcell Wagner,† Wael M. Abdel-Mageed,‡,§ Rainer Ebel,‡ Alan T. Bull,⊥ Michael Goodfellow,∥ Hans-Peter Fiedler,*,† and Marcel Jaspars*,‡ †
Mikrobiologisches Institut, Universität Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Old Aberdeen, Scotland, AB24 3UE, U.K. § Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut, Egypt ⊥ Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, U.K. ∥ School of Biology, University of Newcastle, Newcastle upon Tyne, NE1 7RU, U.K. ‡
S Supporting Information *
ABSTRACT: Dermacoccus abyssi sp. nov. strains MT1.1 and MT1.2 are actinomycetes isolated from a Mariana Trench sediment at a depth of 10 898 m. The fermentation process using complex media led to the production of three new pigmented heteroaromatic (oxidized and reduced) phenazine compounds, dermacozines H−J (1−3). Extensive use was made of 1D and 2D NMR experiments and high-resolution MS to determine the structures of the compounds. The new dermacozines showed radical scavenging activity, and the highest activity was observed for dermacozine H (1), with an IC50 value of 18.8 μM.
P
reviously, 38 novel actinomycete strains were isolated from a sediment sample that was collected by the remotely operated submersible Kaiko (dive no. 74, 1998) at the deepest part of the world’s oceans, namely, the Mariana Trench in the western Pacific Ocean, within which the Challenger Deep, at its southernmost end, is the deepest point on Earth.1 Its depth is variously reported to be 10 915 to 10 920 m.2,3 Deep-sea sediments are of particular interest in view of their microbial diversity4,5 and potential for drug development from marine natural products.6−8 Recently we reported the isolation and characterization of dermacozines A−G, a new family of phenazine compounds that were produced by Dermacoccus abyssi sp. nov., strains MT1.1 and MT1.2.9 Both strains, belonging to the order Actinomycetales, originated from the above-mentioned sediment collected from Challenger Deep at a depth of 10 898 m. Our ongoing studies resulted in the detection and characterization of three additional phenazine compounds, dermacozines H (1), I (2), and J (3). In this second report we describe the fermentation of the strains, isolation and structure determination, and the biological evaluation of the new dermacozines. In addition to dermacozines A−G, several minor congeners were detected in the culture filtrate extract of strain Dermacoccus MT1.2 by HPLC-diode array analysis whose UV−vis spectra indicated their affiliation with the dermacozine family. The scale-up of the cultivation from the 500 mL Erlenmeyer flask scale to a 10 L aerated stirred tank fermentor was hampered by a strong foaming of the culture. The © 2014 American Chemical Society and American Society of Pharmacognosy
optimization of the production medium, doubling the amount of glucose and CaCO3, together with a variation in the preculture handling led to a 2-fold increase of dermacozine production and eliminated foaming of the culture. A combination of 48 h preculture incubation in shake flasks followed by a further 24 h incubation as stationary culture or 24 h incubation in shake flasks followed by 48 h cultivation as stationary culture, respectively, permitted the cultivation of the Received: November 14, 2013 Published: February 5, 2014 416
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by a succession of selective chromatographic steps. Dermacozines H (1), I (2), and J (3) were obtained in amounts of 5.6, 6.6, and 12 mg, respectively, and characterized by spectroscopic methods. Dermacozine H (1) was obtained as an orange-brown powder. The molecular formula was established as C16H13N3O4 by HRESIMS measurement, thus implying 12 degrees of unsaturation. The UV−vis spectrum showed absorption maxima at 215, 297, 421sh, and 459 nm. The 1H and 13C NMR spectra in combination with HSQC NMR data (Table 1) of 1 exhibited signals of one methyl group [H3-16 (δH 2.92)], five aromatic methines in two aromatic spin systems [H-2, H-4, δH 7.68 (brs), 6.40 (brs); H-7, H-8, and H9, δH 6.60 (d, 8.0), 6.51 (t, 8.0), and 6.29 (d, 8.0)], and one aldehyde proton at δH 9.49 (s). Nine quaternary carbon atoms were observed in the 13C NMR spectrum, comprising two carbonyl groups, C-13 and C-11 (δC 170.6 and 168.7), four nitrogenated carbons, C-4a, C-5a, C-9a, and C-10a (δC 137.9, 133.0, 136.0, and 145.7), and three olefinic carbons, C-1, C-3, and C-6 (δC 117.6, 127.3, and 123.8 ppm), in addition to a characteristic aldehyde carbon, C-15 (δC 190.0). Moreover, three hydrogen resonances could be attributed as attached to nitrogen atoms, i.e., NH2-14 (δH 7.71, 7.31) and NH-10 (δH 12.56). Key HMBC correlations included H-2 to C-11 and C10a; H3-16 to C-4a and C-5a; H-9 to C-7 and C-5a; H-7 to C13; NH-10 to C-4a, C-5a, C-9a, and C-1; H-15 to C-3; and H-4 to C-15, thus establishing the position of the aldehyde group at C-3. Correspondingly the 13C NMR spectrum showed downfield shifts for C-3 and C-2 and an upfield shift for C-4 (δC 127.3, 132.8, and 108.5) in comparison to the respective signals in dermacozines A and B.9 The NOE correlations (Supporting Information Figure 7S) from H-15 to H-2 and H4; NH-10 to H-9; H3-16 to H-4 and NH2-14; and NH2-14 to H-7 clearly confirmed the planar structure as that shown in 1. Dermacozine I (2) was obtained as dark pink powder. On the basis of its molecular formula, C22H18N4O3, as established by HRESIMS, 2 was identified as an isomer of the previously reported dermacozine B,9 which was corroborated by the close resemblance in the NMR data of both compounds. The main difference was the presence of two ortho-positioned protons, H7 and H-8 (δH 6.54 and 6.61, each d, 8.0), in the 1H NMR spectrum of 2, instead of two meta-coupled protons in dermacozine B. Key HMBC correlations from H-2 and H-7 to the primary amide carbonyl groups C-11 and C-20, respectively, H-8 to the benzoyl carbonyl group C-13, and from the protons assigned to the N-methyl group H3-22 as well as from NH-10 to C-4a and C-5a established the structure of demacozine I (2) as depicted. Dermacozine J (3) was obtained as a yellow-orange powder. From the HRESIMS spectrum, the molecular formula for the oxidized form of 3 was established as C27H24N5O6S, indicating a molecular formula of C27H25N5O6S for the parent compound. The 1H and 13C NMR spectra in combination with 1H−13C HSQC NMR data (Table 1) of 3 exhibited signals of two methyl groups [H3-22 δH 2.94 (s) for the N-Me and H3-28 δH 1.83 (s) for COCH3]; nine aromatic methines in three aromatic spin systems [H-2, H-4, δH 7.48 (brs), 6.59 (brs); H-7, H-8, δH 6.63 (d, 8.1), 6.79 (d, 8.1)]; phenyl protons [H-15/H-19, δH 7.71 (d, 7.5), H-16/H-18, δH 7.54 (t, 7.5), and H-17 δH 7.63 (t, 7.5)] in addition to one aliphatic methine [H-25 δH 4.12 (m)] and methylene [H2-24 δH 3.13 (dd, 13.2, 3.6) and 2.95 (m)]. The 13C NMR spectrum revealed 14 quaternary carbon atoms comprising five carbonyls, three amides [C-11 (δC 169.9), C-20
strain in fermentors without foaming. Such a fermentation course is shown in Figure 1.
Figure 1. Time course of a 10 L fermentation of Dermacoccus strain MT1.2 in dependence on the preculture handling. (a) Growth measured by the DNA concentration and (b) production of the main component, dermacozine C, are dependent on the type of preculture: (□ or ■) 24 h shake flask followed by 48 h stationary flask cultivation; (○ or ●) 48 h shake flask followed by 24 h stationary flask cultivation; (◇ or ◆) control, 72 h shake flask cultivation.
Previous reports have shown the strong impact of the preculture conditions on the fermentation process on productivity as well as cell growth.10,11 To the best of our knowledge, this is the first study that describes a strong increase of secondary metabolite yields in the fermentation of actinomycetes caused by a mixture of shake flask and stationary flask incubation of the preculture. This effect is not attributed to the growth rate of the producing organism because it was similar to the control, which was uniformly inoculated with shake flask precultures. An explanation of this positive effect can be given by the fact that during stationary cultivation the secondary metabolism is activated due to lack of oxygen supply. It is known that phenazines promote anaerobic survival of Pseudomonas aeruginosa.12 Dermacocci are obligate aerobic microorganisms,13 and oxygen is needed for electron transfer in the respiratory chain. Some phenazines have been reported to act as potent radical scavengers and electron acceptors and possibly substitute the lack of oxygen, especially in their natural deep-sea (oxygen-deprived) habitat.14 This finding is supported by our previous study of the antioxidant activities of the dermacozines, especially of the major compound, dermacozine C.9 Dermacozines were isolated from the culture filtrate by Amberlite XAD-16 chromatography and subsequently purified 417
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Table 1. NMR Spectroscopic Data for Dermacozines H−J (1−3) 1 no.
δC,a type
1 2 3 4 4a 5a 6 7 8 9 9a 10 10a 11 12
117.6, 132.8, 127.3, 108.5, 137.9, 133.0, 123.8, 122.3, 120.9, 113.2, 136.0,
13 14
170.6, C
15 16 17 18 19 20 21
190.0, CH 39.3, CH3
2 δHc (J in Hz)
C CH C CH C C C CH CH CH C
δC,a type 114.8, 121.3, 121.1, 115.8, 138.5, 134.8, 125.4, 120.1, 124.2, 116.9, 141.4,
7.68, brs 6.40, brs
6.60, d (8.0) 6.51, t (8.0) 6.29, d (8.0)
3 δHc (J in Hz)
C CH CH CH C C C CH CH C C
7.02, d (8.0) 6.66, d (8.0) 6.45, d (8.0)
6.54, d (8.0) 6.61, d (8.0)
12.56, s
111.0, 125.8, 128.2, 114.3, 138.1, 133.4, 123.5, 122.6, 126.6, 117.8, 136.8,
δHd (J in Hz)
C CH C CH C C C CH CH C C
7.48, brs 6.59, brs
6.63, d (8.1) 6.79, d (8.1)
11.84, brs
145.7, C 168.7, C
136.5, C 169.7, C
10.97, brs 143.2, C 169.9, C
12-OHe
a. 7.83, brs b. 7.24, brs 195.8, C 139.1, C
a. 7.71, brs b. 7.31, brs 9.49, s 2.92, s
129.2, 128.9, 131.8, 128.9, 129.2, 170.2,
22 24
a. 8.08, brs b. 7.40, brs 193.5, C 137.7, C
CH CH CH CH CH C
7.61, 7.52, 7.61, 7.52, 7.61,
m m m m m
a. 7.83, brs b. 7.45, brs 2.98, s
40.0, CH3
25 26 27 28 29 30 a
δC,b type
129.3, 128.5, 132.0, 128.5, 129.3, 169.9,
CH CH CH CH CH C
7.71, 7.54, 7.63, 7.54, 7.71,
d (7.5) t (7.5) t (7.5) t (7.5) d (7.5)
a. 7.79, brs b. 7.39, brs 2.94, s a. 3.13, dd (13.2, 3.6) b. 2.95, m 4.12, m 7.82, m
39.7, CH3 37.0, CH2 53.0, CH 168.9, C 22.7, CH3 172.2, C
1.83, s 30-OHe
b
c
d
e
At 100 MHz in DMSO-d6. At 150 MHz in DMSO-d6. At 400 MHz in DMSO-d6. At 600 MHz in DMSO-d6. Not observed.
(δC 169.9), and C-27 (δC 168.9)], a ketone [(C-13, δC 193.5 s)], a carboxylic acid [C-29 (δC 172.2)], four nitrogenated carbons [C-4a, C-5a, C-9a, and C-10a (δC 138.1, 133.4, 136.8, and 143.2)], and five sp2 carbons [C-1, C-3, C-6, C-9, and C-14 (δC 111.0, 128.2, 123.5, 117.8, and 137.7)]. From the results of the 1H−15N HSQC experiment, it was evident that six protons were bonded to nitrogen, comprising two NH2 groups [NH2-12 (δH 8.08, 7.40) and NH2-21 (δH 7.79, 7.39)] and two NH groups [NH-10 (δH 10.97) and NH26 (δH 7.82)]. From the 1H−15N HMBC NMR spectrum of 3 it was possible to assign the resonance of each nitrogen: NH212 (δN 106.8), NH-21 (δN 111.3), NH-10 (δN 99.6), NH-5 (δN 121.2), and NH-25 (δN 123.6) (Figure 2a). With all protons assigned to their directly attached carbon and nitrogen atoms, it was possible to deduce eight substructures, and the connectivities between these substructures were established from key HMBC correlations (Figure 2b). Thus, correlations from H-2 to C-11; H-2 and H-4 to C10a; H-7 to C-20; H-8 to C-6; and H3-22 to C-4a and C-5a clearly defined the positions of the amide and methyl groups. The position of the benzoyl moiety on C-3 was defined from
Figure 2. (a) 15N NMR data for 3 (H→N). (b) Observed COSY correlations (bold bonds) and long-range HMBC (H → C) correlations for 3.
HMBC correlations from H-2, H-4, and H-15/H-19 to C-13. Correspondingly the 13C NMR spectrum showed downfield shifts for C-3 and C-2 and an upfield shift for C-4 (δC 128.2, 125.8, and 114.3) in comparison to the respective signals in dermacozines A and B.9 Also, HMBC correlations from NH-10 to C-4a, C-5a, C-1, and C-9 confirmed the reduced phenazine skeleton. Further HMBC correlations from H-24 to C-9 and C29; from H-25 to C-24, C-27, and C-29; from NH-26 to C-27 418
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were discarded, and the supernatant (8.5 L) was applied to an Amberlite XAD-16 column (35 × 5.5 cm; Rohm and Haas). Dermacozines were eluted with H 2 O−MeOH (40:60). The dermacozine-containing fractions were concentrated in vacuo to an aqueous residue and extracted five to 10 times with EtOAc at pH 4. The organic extracts were concentrated in vacuo, dissolved in CH2Cl2, and applied to a diol-modified silica gel column (40 × 2.6 cm). The separation was carried out by linear CH2Cl2−MeOH gradient elution. Further purification of discrete dermacozine-containing fractions was done using Sephadex LH-20 and Toyopearl HW40-F column chromatography (90 × 2.5 cm). Pure dermacozines were obtained by preparative reversed-phase HPLC (Jupiter 4 μ Proteo 90 Å, 250 × 10 mm, 4 μm) using a gradient of 0−90% CH3CN−H2O over 40 min. Dermacozine H (1) was obtained in an amount of 5.6 mg, dermacozine I (2) 6.6 mg, and dermacozine J (3) 12 mg. HPLC-Diode Array Analysis. The HPLC analyses, database evaluation by an in-house HPLC-UV−vis database that contained 950 reference compounds, and sample preparation were performed as described earlier.17,18 Dermacozine H (1): orange-brown powder, 5.6 mg; UV (EtOH) λmax (log ε) 459 (3.4), 421sh (3.3), 297 (4.0), 215 (4.2) nm; 1H and 13 C NMR data (DMSO-d6), see Table 1; HRESIMS [M]+ m/z 311.0901 (calcd for C16H13N3O4, 311.0906; Δ = −1.6 ppm). Dermacozine I (2): dark pink powder, 6.6 mg; UV (EtOH) λmax (log ε) 516 (3.5), 379 (3.1), 314 (3.3), 263 (4.2) nm; 1H and 13C NMR data (DMSO-d6), see Table 1; LRESIMS [M + H]+ m/z 387.0, [M − H]− m/z 385.0; HRESIMS [M + H]+ m/z 387.1455 (calcd for C22H19N4O3, 387.1457; Δ = −0.5 ppm). Dermacozine J (3): yellow-orange powder, 12 mg; UV (EtOH) λmax (log ε) 435 (3.6), 297 (3.9), 249 (4.0), 215 (4.1) nm; 1H and 13C NMR data (DMSO-d6), see Table 1; HRESIMS [M]+ m/z 546.1429 (calcd for C27H24N5O6S, 546.1442; Δ = −2.4 ppm). DPPH Radical Scavenging Assay. The determination of the antioxidant activities of the dermacozines was performed in a DPPH radical scavenging assay as reported by Abdel-Mageed et al.9,19,20
and C-29; and from H3-28 to C-27 indicated an Nacetylcysteine moiety attached to C-9. Correspondingly the 13 C NMR spectrum showed downfield shifts for C-9 and C-8 (δC 117.8 and 126.6) in comparison to the respective signals in 1 (δC 113.2 and 120.9) as well as in dermacozine B.9 The apparent structural difference between 3 and dermacozine B9 is the N-acetylcysteine moiety covalently attached to C-9 of the phenazine backbone via the nucleophilic thiol group. Also, there is a good agreement between literature15 and observed chemical shifts for this group. N-Acetylcysteine adducts have been reported earlier with the phenazine compounds16 as well as macrocyclic peptides15 produced by Streptomyces species. In our previous publication dermacozines were shown to exhibit cytotoxic and radical scavenging activities.9 As dermacozine C displayed the strongest radical scavenging activity (IC50 = 8.4 μM), superior to that of ascorbic acid (IC50 = 12.1 μM), it was used as a reference in Table 2 to estimate Table 2. DPPH Radical Scavenging Activity IC50 (μM) dermacozine C dermacozine H dermacozine I dermacozine J ascorbic acid
8.4 18.8 34.6 19.6 12.1
the activities of the isolated novel dermacozine compounds. Dermacozine H (1) showed the next best radical scavenging activity (IC50 = 18.8 μM), followed by dermacozine J (3) (IC50 = 19.6 μM), and finally dermacozine I (2) (IC50 = 34.6 μM) (Table 2).
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EXPERIMENTAL SECTION
ASSOCIATED CONTENT
S Supporting Information *
General Experimental Procedures. UV spectra were obtained using a UV/visible light spectrophotometer (Perkin-Elmer Instruments, Lambda 25 (UV/vis) spectrometer). 1H, 13C, and all NMR 2D experiments were recorded on a Varian Unity VNMRS 600 and 400 MHz spectrophotometer, in DMSO-d6. A low-resolution electrospray mass spectrum was obtained using a Perseptive Biosystems Mariner LC-MS, and high-resolution mass data were obtained on a Finnigan MAT 900 XLT. High-resolution mass spectrometric data were obtained from a Thermo Instruments MS system (LTQ XL/LTQ Orbitrap Discovery) coupled to a Thermo Instruments HPLC system (Accela PDA detector, Accela PDA autosampler, and Accela pump). The following conditions were used: capillary voltage 45 V, capillary temperature 320 °C, auxiliary gas flow rate 10−20 arbitrary units, sheath gas flow rate 40−50 arbitrary units, spray voltage 4.5 kV, mass range 100−2000 amu (maximum resolution 30 000). Analytical HPLC analyses were carried out with an HP 1090 liquid chromatograph, diode-array detector, and HP Kayak XM 600 ChemStation (Agilent) and an Agilent 1200 series gradient pump, diode array detector, and 6330 ion trap LC-MS (Agilent). Microorganisms. Dermacoccus abyssi strains MT1.1 and MT1.2 were isolated previously1 and provided by the School of Biology, University of Newcastle, UK. Fermentation and Isolation. For isolation of 1−3, batch fermentations were carried out with Dermacoccus strain MT1.2 in a 10 L stirred tank fermentor using a complex medium that consisted of glucose 20 g, glycerol 10 g, oatmeal 5 g, yeast extract 5 g, Bacto casamino acids 5 g, and CaCO3 2 g in 1 L of tap water, adjusted to pH 7.0. The fermentor was incubated at 27 °C, an agitation of 250 rpm, and aeration rate of 0.5 volume air per volume per min. The highest yields of dermacozines and DNA were measured at 192 h of fermentation. Dermacozine C, the main compound, reached a maximal yield of 50 mg L−1. The fermentation broth was centrifuged, the cells
UV, 1D and 2D NMR, and MS spectra of compounds 1−3 are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(H.-P. Fiedler) E-mail: hans-peter.fi
[email protected]. Fax: +49 7071 295999. Tel: +49 7071 2972079. *(M. Jaspars) E-mail:
[email protected]. Fax: +44 1244272921. Tel: +44 1224 272895. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful to the Kaiko operation team and the crew of M.S. Yokosuka, JAMSTEC, Yokosuka, Japan, for collecting sediment samples. M.W. thanks the Jürgen-ManchotFoundation for a generous scholarship and Max-BuchnerForschungsstiftung for support of consumables. M.J. is the recipient of a BBSRC Research Development Fellowship. The authors thank Mr A. Kulik, University of Tübingen, Germany, for assistance in fermentation and HPLC-ESIMS analysis. The authors are grateful to EPSRC National Mass Spectrometric Centre, University of Wales, Swansea, for mass spectrometric analysis.
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REFERENCES
(1) Pathom-aree, W.; Stach, J. E. M.; Ward, A. C.; Horikoshi, K.; Bull, A. T.; Goodfellow, M. Extremophiles 2006, 10, 181−189.
419
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Journal of Natural Products
Note
(2) Taira, K.; Kitagawa, S.; Yamashiro, T.; Yanagimoto, D. J. Oceanogr. 2004, 60, 919−926. (3) Gvirtzman, Z.; Stern, R. J. Tectonics 2004, 23, (TC2011) 10.1029/2003TC001581. (4) Stach, J. E. M.; Maldonado, L. A.; Masson, D. G.; Ward, A. C.; Goodfellow, M.; Bull, A. T. Appl. Environ. Microbiol. 2003, 69, 6189− 6200. (5) Maldonado, L. A.; Stach, J. E. M.; Pathom-aree, W.; Ward, A. C.; Bull, A. T.; Goodfellow, M. Antonie van Leeuwenhoeck 2005, 87, 11− 18. (6) Bull, A. T.; Stach, J. E. M. Trends Microbiol. 2007, 15, 491−499. (7) Williams, P. G. Trends Biotechnol. 2008, 27, 45−52. (8) Molinski, T. F.; Dalisy, D. S.; Lievens, S. L.; Saludes, J. P. Nat. Rev. 2009, 8, 69−85. (9) Abdel-Mageed, W. M.; Milne, B. F.; Wagner, M.; Schumacher, M.; Sandor, P.; Pathom-aree, W.; Goodfellow, M.; Bull, A. T.; Horikoshi, K.; Ebel, R.; Diederich, M.; Fiedler, H.-P.; Jaspars, M. Org. Biomol. Chem. 2010, 8, 2352−2362. (10) Neves, A. A.; Vieira, L. M.; Menezes, C. J. Biotechnol. Bioeng. 2000, 72, 628−633. (11) Hornbäk, T.; Nielsen, A. K.; Dynesen, J.; Jakobsen, M. FEMS Microbiol. Lett. 2004, 236, 145−151. (12) Wang, Y.; Kern, S. E.; Newman, D. K. J. Bacteriol. 2010, 192, 365−369. (13) Stackebrandt, E.; Koch, C.; Gvozdiak, O.; Schumann, P. Int. J. Syst. Bacteriol. 1995, 45, 682−692. (14) Blokhina, O.; Virolainen, E.; Fagerstedt, K. V. Ann. Bot. 2003, 91, 179−194. (15) Schulz, D.; Nachtigall, J.; Riedlinger, J.; Schneider, K.; Poralla, K.; Imhoff, J. F.; Beil, W.; Nicholson, G.; Fiedler, H.-P.; Süssmuth, R. D. J. Antibiot. 2009, 62, 513−518. (16) Gilpin, M. L.; Fulston, M.; Payne, D.; Cramp, R.; Hood, I. J. Antibiot. 1995, 48, 1081−1085. (17) Fiedler, H.-P. Nat. Prod. Lett. 1993, 2, 119−128. (18) Brötz, E.; Kulik, A.; Vikineswary, S.; Lim, C.-T.; Tan, G. Y. A.; Zinecker, H.; Imhoff, J. F.; Paululat, T.; Fiedler, H.-P. J. Antibiot. 2011, 64, 257−266. (19) Ibraheim, Z. Z.; Abdel-Mageed, W. A.; Dai, H.; Guo, H.; Zhang, L.; Jaspars, M. Phytother. Res. 2012, 26, 579−586. (20) Abdel-Mageed, W. M.; Backheet, E. Y.; Khalifa, A. A.; Ibraheim, Z. Z.; Ross, S. A. Fitoterapia 2012, 83, 500−507.
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