Note pubs.acs.org/jnp
Insights into Structure−Activity Relationships of Bacterial RNA Polymerase Inhibiting Corallopyronin Derivatives Till F. Schab̈ erle,† Alexander Schmitz,† Georg Zocher,‡ Andrea Schiefer,§ Stefan Kehraus,† Edith Neu,† Martin Roth,⊥ Dmitry G. Vassylyev,∥ Thilo Stehle,‡ Gabriele Bierbaum,§ Achim Hoerauf,§ Kenneth Pfarr,§ and Gabriele M. König*,† †
Institute for Pharmaceutical Biology, University of Bonn, Bonn, Germany Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany § Institute for Medical Microbiology, Immunology and Parasitology, University Hospital Bonn, Bonn, Germany ⊥ Leibniz Institute for Natural Product Research and Infection Biology e.V., Hans Knöll Institute, Jena, Germany ∥ Department of Biochemistry and Molecular Biology, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama, United States ‡
S Supporting Information *
ABSTRACT: The new compound precorallopyronin A is a stable precursor in the biosynthesis of the antibiotic corallopyronin A. This natural product was isolated from the producer strain Corallococcus coralloides B035. Together with various semisynthetically obtained corallopyronin A derivatives its antibacterial effects were evaluated. In combination with an X-ray crystallization model limitations of derivatization possibilities were revealed. The antibiotic potential of the novel precorallopyronin A is comparable to that of the structurally more complex corallopyronin A, which highlights that the additional chiral center is not essential for activity.
D
membranes (worm cell, vesicle, Wolbachia inner and outer membranes) that the antibiotic has to cross to reach its target inside the symbiotic bacteria. It was shown for myxopyronin A (6) that changes in the right part of the molecule (which is identical with that of corallopyronin A, colored in red in Figure 1) diminish antibacterial effects. This part of the molecule interacts with the target RNAP in multiple ways, and thus structural changes are detrimental.7 The superior antibiotic activity of corallopyronin A in comparison to myxopyronin A was suggested to be due to the longer lipophilic western chain of corallopyronin A, which occupies an extra binding pocket in the RNAP. The biological effect from further changes in the western side chain of these pyrone antibiotics has not been reported. We have applied two strategies to obtain analogues with a modified western chain, i.e., the search for natural analogues and the semisynthetic modification of corallopyronin A. C. coralloides B035 was cultivated with the addition of an adsorber resin. This resin was eluted with acetone, and the resulting extract used for the isolation and subsequent structure elucidation of a new natural product with promising antibiotic activity. The previously described metabolite corallopyronin A (1) was isolated together with a new analogue, precorallopyr-
ue to the increasing number of multi- and pan-antibioticresistant bacteria, there is an urgent need for new antibiotics.1 Myxobacteria are a rich source of antibiotics,2−4 and one of the most promising is corallopyronin A (1).5 This in vivo active antibiotic is currently the subject of preclinical studies for further development. Isolated from Corallococcus coralloides strains, three analogues are known so far, i.e., corallopyronin B (2), corallopyronin A′ (3), and corallopyronin C (4). In an anti-infective assay utilizing several bacteria, corallopyronin A′ showed good activity against Gram-positive bacteria with MIC values in the range of 0.1 μg mL−1 (Staphylococcus aureus) and 0.8 μg mL−1 (Bacillus megaterium).5,6 Corallopyronins A′, B, and C were less active in the same test systems, with MIC values in the range of 0.4−0.8 μg mL−1 (S. aureus) and 1.6−6.3 μg mL−1 (B. megaterium). Later it was shown that corallopyronin A is a specific inhibitor of bacterial DNA-dependent RNA-polymerase (RNAP), but shows no cross-resistance with rifampicin, another RNAP inhibitor, due to its unique binding site deep inside the RNAP clamp head domain.7,8 In addition to the antibacterial effect, in vivo antifilarial activity was found by treating infected BALB/c mice with an effective concentration comparable to doxycycline.9 This is due to the depletion of intracellular Wolbachia, obligate intracellular endosymbiotic bacteria of worms. The observed activity is extraordinary, considering the many intervening surfaces (blood vessels, pleura, worm cuticle) and © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 23, 2015
A
DOI: 10.1021/acs.jnatprod.5b00175 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 1. Structures of corallopyronin derivatives corallopyronin A (1), corallopyronin B (2), corallopyronin A′ (3), corallopyronin C (4), precorallopyronin A (5), myxopyronin A (6), desmethyl myxopyronin B (7), and methyl-N-(6-[sphingosyl-N-carbonyl]hexyl)carbamate (8). The two different polyketide chains that are merged in the biosynthesis to yield the pyrone ring are color coded. The red side chain is referred to in the text as the eastern chain, and the black one as the western chain, respectively. For 5 the carbon atoms are numbered as applied in the text.
a chemical shift of δ 32.2 instead of δ 69.6 as in corallopyronin A. This confirmed the loss of the C-24 hydroxy group in the new molecule. The 13C NMR resonances of carbon atoms in the neighborhood of C-24 have resonance signals shifted by 2− 7 ppm in comparison to the corallopyronin A spectrum. Also, methyl groups C-26 resonating at δ 23.6 and C-22 at δ 41.5 showed some differences when compared to corallopyronin A (see Table S1). On the basis of the biosynthetic gene cluster a double bond was initially suggested between C-24 and C-25.6 However, the 13C NMR chemical shift of C-24 does not correspond with a sp-hybridized carbon. At the same time, in 5 C-27 was attributed to a 13C NMR resonance at δ 124.7, indicating a double bond between C-25 and C-27, which was proven by COSY and HMBC correlations (see Table 1). The 13 C NMR shift of C-26 (δ 23.6) indicated that the double bond Δ25,27 is Z-configured.6 The specific rotation of compound 5 has the same direction as that of myxopyronin A (6) [−73.5 in MeOH (c = 0.3)].7 This indicates that C-7, the only asymmetric carbon atom in the molecule, is R-configured, as determined for 6 and 1.6 We propose the trivial name precorallopyronin A (5 in Figure 1). Our finding gives an indication of the timing of the post-PKS modification reactions. The PKS-derived western chain of corallopyronin A suggests a carbon−carbon double bond between C-24 and C-25, as such double bonds are typically incorporated between acetate building blocks. In compound 5 the double bond is between C-25 and C-27, suggesting that an isomerization reaction takes place before the introduction of the hydroxy group (Supporting Information, Figure S1). Precorallopyronin A (5) was considered an ideal tool to obtain insights into structure−activity relationships of the corallopyronins. The antibacterial activities of precorallopyronin A (5) were found to be similar to those of corallopyronin A (1), with only a slight elevation of MIC values (Table 2). In vitro assays assessing activity against Wolbachia cultured in insect cells revealed that the structurally simpler precorallopyronin A (5) has an antibiotic effect comparable to that of corallopyronin A (Figure 2). The ratio of Wolbachia 16S rRNA copies to C6/36 actin copies, the latter from the insect cells, was strongly decreased in experiments starting with 0.01 μg mL−1 precorallopyronin A
onin A (5), most probably a precursor in the biosynthesis of corallopyronin A missing the hydroxy group at C-24 that was obtained in minor amounts. The hydroxy group at carbon C-24 of the western chain of corallopyronin A was suspected to be a good anchor for chemical reactions, and modifications at this position may improve the binding at the target site via additional interactions. Indeed, in their original paper on the crystal structure of RNAP in complex with the related myxopyronin (6) the authors indicated additional space in the adjacent hydrophobic pocket for functionalities at C-24.7 Recently it was reported that mutations conferring resistance to corallopyronin A arise, but the frequency was lower than that to rifampicin, an RNAP inhibitor in clinical use.9 Therefore, derivatives of corallopyronin A would be of interest in light of the rapid resistance development against RNAP inhibitors. Deciphering the corallopyronin A biosynthetic gene cluster in C. coralloides B035 suggested precorallopyronin could be an intermediate.6 A large-scale fermentation provided material from which this new corallopyronin-like compound was isolated. The 1H NMR spectrum of compound 5 is very similar to that of corallopyronin A. Many characteristic signals were detectable in the 1H NMR spectrum, especially the two doublet resonances for H-12 and H-19 at δ 6.44 and δ 6.27, respectively (Table 1). The singlet resonance of the pyronering proton H-5 at δ 5.80 and the carbamate methyl group protons H3-14, which resonate at δ 3.71, were observable. The multiplets of methine and methylene group protons H-29, H30, and H2-28 at δ 5.42, 5.44, and 2.70, respectively, were discernible as well as protons H-11 and H-7 at δ 5.10 and 2.56, respectively. The main differences when compared with corallopyronin A were the upfield shifts of the resonance signal for H-27 and H2-24, the protons next to the hydroxy group in corallopyronin A. Several additional resonances in the range of 1 to 2 ppm evidenced that the isolated compound is a new corallopyronin derivative. The high-resolution mass was found to be at m/z 534.2826 [M + Na]+, which corresponds to the sodium adduct of a molecule with the formula C30H41NO6. Most 13C NMR signals of the new compound were in agreement with those of corallopyronin A, except six signals from carbons close to C-24 of corallopyronin A. The highest deviation was found for the 13C NMR signal of C-24 itself with B
DOI: 10.1021/acs.jnatprod.5b00175 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. NMR Spectroscopic Data of Precorallopyronin A (5) in MeOH-d4 (1H, 300 MHz; 13C, 75 MHz) corallopyronin A (1)
precorallopyronin A (5) no.
δC, mult
2 3 4 5 6 7 8 9
a
10 11 12
28.6, CH2 110.9, CH 125.6, CH
13 14 15 16 17 18 19
156.8, C 52.6, CH3 201.7, C 136.5, C 11.9, CH3 136.6, CH 122.8, CH
20 21 22
149.3, C 17.2, CH3 41.5, CH2
23 24
,C 102.7, C a ,C 107.5, CH 168.9, C 38.7, CH 18.6, CH3 35.9, CH2
δH, mult (J in Hz)
COSY
δC
8 7
8, 9, 10 4, 6, 7 7, 10, 11
168.1 102.7 181.3 107.7 168.4 38.7 18.7 36.0
11 10, 12 11
7, 9, 11 12 10, 11, 13
28.5 111.0 125.7
5.80, s 2.56, m 1.25, d (6.7) 1.56, m, 1.79 m 2.04, m 5.10 m 6.44, d (14.8)
HMBC (H to C)
7
3.71, s
13
1.95, s 7.29, m 6.27, br d (11.1)
15, 16, 18
27.3, CH2 32.2, CH2
1.83, s 2.19, br t (6.7) 1.61, m 2.08, m
19, 20, 22 19, 21, 23, 24 22, 24 22, 23, 25, 26
25 26 27 28
136.6, C 23.6, CH3 124.7, CH 32.0, CH2
1.74, s 5.19, m 2.70, m
29 30 31
131.3, CH 125.7, CH 18.1, CH3
5.42, m 5.44, m 1.67, d (4.5)
21, 23 22, 24
27 26, 28 26, 27, 29, 30 28 31 30
Figure 2. Precorallopyronin A (Pre-CorA) and corallopyronin A (CorA) deplete Wolbachia from C6/36 insect cells. A concentrationdependent loss of Wolbachia can be observed due to the inclusion of these antibiotics in the medium. Precorallopyronin A at 0.1 μg/mL resulted in a loss of Wolbachia comparable to doxycycline. The ratios of Wolbachia 16S rRNA copies/μL normalized to C6/36 actin copies/ μL as determined by qPCR are given.
corallopyronin A (1) when compared to precorallopyronin A (5). It can be concluded that the OH group at C-24 minimally enhances the activity of the antibiotic corallopyronin A. Thus, precorallopyronin A is a further good candidate for an antiWolbachia agent, even though in vivo data are currently missing because of the low supply. A putative role of the hydroxy group in the pharmacokinetics of corallopyronin A has to be analyzed in the future. To obtain further insights into the structure−activity relationship of corallopyronin A, several analogues were made. Changes in the eastern chain were expected to diminish antibacterial effects because this part of the molecule is interacting with the target RNAP in multiple ways.8 The hydroxy group at C-24 seemed an ideal site for modification, e.g., acylation reactions and oxidation. The antibiotic activity of all derivatives is shown in Table 3 (details concerning the semisynthesis and the resulting structures can be found in the Supporting Information). None of the derivatives had a better activity than corallopyronin A (1) itself. In general, modifications at C-24 led to less active or inactive molecules. The dramatic decrease
156.8 52.6 201.3 136.5 11.7 137.6 122.8 148.8 17.4 37.9 34.3 69.6
24, 25, 27 26, 28, 29 25, 29, 30
138.5 17.7 126.4 31.4
28, 31 28 29, 30
130.9 126.1 18.1
Table 3. MIC Values [μg mL−1] of Corallopyronin A (1) and Its Semisynthetic Derivatives against Several Bacteriaa
a13
C NMR signals not detectable.
Table 2. MIC Values [μg mL−1] of Corallopyronin A (1) and Precorallopyronin A (5) organism
corallopyronin A
precorallopyronin A
Bacillus cereus Bacillus licheniformis DSM13 Bacillus subtilis 168 Micrococcus luteus ATCC 4698 Mycobacterium smegmatis Staphylococcus aureus SG 511 Staphylococcus simulans 22
2 2 16 1 >64 0.5 1
2 4 32 4 >64 2 4
compound corallopyronin A acetyl-corallopyronin A decanoyl-corallopyronin A acetoxyacetyl-corallopyronin A furoyl-corallopyronin A benzoyl-corallopyronin A fluorobenzoyl-corallopyronin A thiophen-2-carboxylcorallopyronin A (S)-MTPAc-corallopyronin A (R)-MTPAc-corallopyronin A oxo-corallopyronin A
compared to medium and vehicle (DMSO) control. Treatment with 0.1 and 0.5 μg mL−1 precorallopyronin A (5) resulted in an even more pronounced effect than 4 μg mL−1 of doxycycline. These results indicated Wolbachia depletion by precorallopyronin A (5). For comparison, corallopyronin A (1) was also assayed at the same time, and a concentrationdependent depletion of Wolbachia was observed (Figure 2). Again, an only slightly more potent activity was seen for
Staph. simulans
M. phlei
B. licheniformis
2 64 >64 >64 64 >64 >64 >64
>64 >64 n.m.b n.m. >64 >64 n.m. n.m.
64 64 64 >64 64 64 >64 >64
64 32 16−32
>64 >64 n.m.
>16d >16d 32−64
a Staphylococcus simulans 22, Mycobacterium phlei (strain from institute collection of IMMIP, University of Bonn, Germany), Bacillus licheniformis MW3. All derivatives are modified at C-24 of 1 (structures in Supporting Information). bn.m.: not measured. cMTPA = αmethoxy-α-trifluoromethylphenylacetic acid. dSupply allowed only tests to an MIC value of 16 μg mL−1.
C
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Figure 3. Structural basis of transcription inhibition by 1 and 5. Middle: The antibiotic (magenta) bound to the hinge-region of the RNAP. Left: Detail of the binding region complexed with precorallopyronin A (5). Right: Detail of the binding region complexed with corallopyronin A (1). The amino acid residues in close contact to the α-pyrone antibiotics are labeled. The model illustrates that the molecules adopt close to identical conformations to fill the tight binding pocket. Only minor variations in the conformation of the tail of the western chain exist.
of the bulkiness of the western chain, as seen in the acylated derivatives, or any changes that increase the rigidity of this chain will abolish antibiotic activity. In conclusion, corallopyronin A and precorallopyronin A are promising antibacterial compounds for further development. They show activity against intracellular bacteria. In this study the limitations in modification of the structures were shown. Every volume gain of the molecules resulted in partial repulsion from the tight binding pocket of the RNAP target. Therefore, if at all, only minor modifications can be taken into account to pursue with the development of α-pyrone antibiotics for clinical use.
in antibiotic activity (∼10-fold) of the compound carrying a ketone instead of a hydroxy functionality at C-24 might be due to a changed 3D structure of the lipophilic chain of the molecule. The rotation of the beforehand sp3-hybridized C-24 is greatly hindered, and the spatial arrangement of the molecule in this region is different. Thereby, it might not fit into the tight binding pocket any more. In a next step a simplified corallopyronin molecule was constructed to test if the central pyrone ring is necessary for the antibiotic activity. In the synthesis of the corallopyronin A analogue, D-sphingosine served as a replacement for the western chain and a seven-membered amino acid was the main building block for the eastern chain (Figure 1 and Figure S2 in the Supporting Information). Reaction of methylchloroformate with aminoheptanoic acid in a dioxane/NaOH mixture resulted in 7-(methoxycarbonyl)aminoheptanoic acid (yield 67%). The product was purified via RP18-HPLC and identified by 1D NMR spectroscopy and LC/MS measurements (details in the Supporting Information). This acid was esterified with D-sphingosine, resulting in the targeted methylN-(6-[sphingosyl-N-carbonyl]hexyl)carbamate (8, yield of 42%) (details in the Supporting Information). The compound had no activity against the Gram-positive bacterium B. megaterium, the Gram-negative bacterium E. coli, or Wolbachia (Supporting Information, Figure S3). Hence, it can be concluded that the pyrone ring is an important feature for the correct arrangement of corallopyronin A in its binding pocket. To rationalize the antimicrobial effects, in silico analyses of corallopyronin A and precorallopyronin A in the RNAP binding pocket were performed. Wolbachia RNAP was modeled based on previous work with a desmethyl derivative of myxopyronin B (7)8 (Figure 3). Our data show that compound 5 fits into the same pocket as corallopyronin A, although a distinctly different conformation of the tail of the western chain is proposed. This means that the central pyrone ring in corallopyronin A and 5 is positioned in the same way, resulting in the formation of the same H-bonds, stabilizing the molecule in the hydrophobic binding pocket. The western chain occupies the adjacent hydrophobic pocket not filled by the myxopyronin derivatives 6 and 7. It is suggested that the long western tail of the corallopyronins has to be flexible to enter and completely occupy the binding pocket. It is also obvious that any increase
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured with a Jasco DIP 140 polarimeter. UV and IR spectra were obtained employing PerkinElmer Lambda 40 and PerkinElmer Spectrum BX instruments, respectively. 1H, 13C, COSY, HSQC, and HMBC NMR spectra were recorded in MeOH-d4 using a Bruker Avance 300 DPX spectrometer operating at 300 MHz for proton and at 75 MHz for 13C, respectively. Spectra were calibrated to residual solvent signals with resonances at δH/C 3.35/49.0 (MeOH-d4). HPLC was performed on a Merck−Hitachi system equipped with an L-6200A pump, an L-4500 A photodiode array detector, a D-6000A interface with D-7000 HSM software, and a Rheodyne 7725i injection system. LRESIMS measurements were performed employing an API 2000, Triple Quadrupole LC/MS/MS (Applied Biosystems/MDS Sciex) with ESI source. HRESIMS were recorded on a Bruker Daltonik micrOTOF-Q time-of-flight mass spectrometer with ESI source. Cultivation, Extraction, and Isolation. Corallococcus coralloides B035 was isolated from a soil sample from Remonchamps, Belgium, in 2001. The strain is stored as a cryo-culture at −80 °C. For the isolation of corallopyronin A and precorallopyronin A the myxobacterium was cultivated in 5 L Erlenmeyer flasks with 1.5 L of MD1 + G medium [3.0 g L−1 casitone, 0.7 g L−1 CaCl2(H2O)2, 2.0 g L−1 MgSO4(H2O)7, and 2.2 g L−1 glucose]. The adsorber resin Amberlite XAD 16 was added in a concentration of 2% to each flask to avoid end product inhibition. For 40 L cultivation 28 flasks were prepared. The flasks were inoculated with precultures (300 mL flasks each with 100 mL of MD1 + G medium) and were cultivated for 7 days at 30 °C and 140 rpm on an HT Orbitron rotary shaker (Infors, Bottmingen, Switzerland) under light-free conditions. Cells and Amberlite XAD 16 were separated from the medium by filtration with a glass filter size 2. Cells and Amberlite XAD 16 were further processed. For desorption of the compounds the adsorber resin (including the bacterial pellet) was placed into a glass filter size 2 and extracted with 6 D
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× 500 mL of acetone (for a 40 L culture). The organic phase was evaporated to result in approximately 25 g of extract, resolved in 250 mL of water, and extracted with 5 × 250 mL of ethyl acetate. The ethyl acetate extract (approximately 4 g) was evaporated and fractionated with RP18-VLC (Polygoprep 60-50 RP18 silica gel (Macherey-Nagel, Düren, Germany). For the fractionation of the ethyl acetate extract a 6.5 cm diameter column was used. The sorbent was poured as a methanolic suspension into the column and compressed under vacuum. Afterward the system was equilibrated with 100 mL of the first eluent, methanol/water (20−80). The sample was dissolved in a small volume of the same solvent mixture and applied onto the top of the column. Nine fractions were obtained through elution with nine different methanol/water mixtures beginning with methanol/water in a ratio of 20:80. In each step the methanol content was increased by 10%, up to 100% methanol for the ninth fraction. It was eluted with 100 mL of solvent for each fraction, except for fraction 9 (100% methanol), where 200 mL was used. VLC fractions 6 eluting with 70% methanol (containing corallopyronin A) and 7 eluting with 80% methanol (containing precorallopyronin A) were further purified via HPLC. VLC 7 was fractionated via RP18-VLC with MeOH/H2O mixtures, getting eight fractions (7.1 to 7.8). Fraction 7.6 was subsequently purified with RP18-HPLC, and the compound eluting at 8−10 min collected. After a second HPLC purification, this time with an acidic modifier (acetic acid), a pure compound was obtained and analyzed via NMR and MS measurements. Precorallopyronin A: yellow oil (1.4 mg); [α]20D −20.6 (c 0.17 MeOH); UV (MeOH) λmax (log ε) 206 (5.42), 289 (4.96) nm; IR (ATR) νmax 2924, 1678, 1573, 1423, 1377, 1250, 1052 cm−1; HRESIMS m/z 534.2826 [M + Na]+ (calcd for C30H41NO6Na m/z 534.2832). MIC Measurement. The tests were performed in polystyrol microtiter plates (Greiner, Kremsmünster, Austria). Corallopyronin A and derivatives were dissolved in a concentration of 1 mg mL−1 in 50% ethanol (stock solution), and dilution series were prepared in microtiter plates through dilution of a stock solution with MüllerHinton medium (Oxoid, Hampshire, UK). The indicator organisms were cultivated on Columbia agar at 37 °C. The day before testing, the organism was transferred into 5 mL of Müller-Hinton medium in 50 mL Erlenmeyer flasks and incubated at 37 °C and 170 rpm on a rotary shaker. A 5 mL amount of new Müller-Hinton medium was incubated the next day with 50 μL of preculture and cultivated to an OD600 of 1.0. To every well of the dilution series was added 100 μL of this solution with 105 cells/mL and then cultivated at 37 °C. MIC values were determined after 24 h. In Vitro Experiments against Wolbachia. In vitro tests were performed with the Aedes albopictus cell line C6/36 infected with Wolbachia from A. albopictus B. It was cultured for 9 days in 96-well plates at 26 °C in L15 Leibovitz’s medium (Invitrogen, Darmstadt, Germany) with and without the different antibiotics [4 μg mL−1 doxycycline hyclate (Merck, Darmstadt, Germany); 1, 0.5, 0.1, 0.05, and 0.01 μg mL−1 corallopyronin A or precorallopyronin A]. Extraction of genomic DNA was performed with the QIAamp kit (Qiagen) according to the manufacturer’s instructions. Depletion of Wolbachia was monitored by quantitative real-time polymerase chain reaction (qPCR) using primers targeting the 16S-rRNA gene of Wolbachia and the A. albopictus B actin gene.10
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Note
AUTHOR INFORMATION
Corresponding Author
*E-mail (G. M. König):
[email protected]. Tel: +49 228 73-3747. Fax: +49 228 73-3250. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support came from the German Centre for Infection Research (DZIF) and from the German Research Foundation (DFG research unit 854).
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REFERENCES
(1) Schäberle, T. F.; Hack, I. M. Trends Microbiol. 2014, 22, 165− 167. (2) Schäberle, T. F.; Lohr, F.; Schmitz, A.; König, G. M. Nat. Prod. Rep. 2014, 31, 953−972. (3) Felder, S.; Dreisigacker, S.; Kehraus, S.; Neu, E.; Bierbaum, G.; Menche, D.; Schäberle, T. F.; König, G. M. Chem. - Eur. J. 2013, 19, 9319−9324. (4) Felder, S.; Kehraus, S.; Neu, E.; Bierbaum, G.; Schäberle, T. F.; König, G. M. ChemBioChem 2013, 14, 1363−1371. (5) Irschik, H.; Jansen, R.; Höfle, G.; Gerth, K.; Reichenbach, H. J. Antibiot. 1985, 38, 145−152. (6) Erol, O.; Schäberle, T. F.; Schmitz, A.; Rachid, S.; Gurgui, C.; El Omari, M.; Lohr, F.; Kehraus, S.; Piel, J.; Müller, R.; König, G. M. ChemBioChem 2010, 11, 1253−1265. (7) Mukhopadhyay, J.; Das, K.; Ismail, S.; Koppstein, D.; Jang, M.; Hudson, B.; Sarafianos, S.; Tuske, S.; Patel, J.; Jansen, R.; Irschik, H.; Arnold, E.; Ebright, R. H. Cell 2008, 135, 295−307. (8) Belogurov, G. A.; Vassylyeva, M. N.; Sevostyanova, A.; Appleman, J. R.; Xiang, A. X.; Lira, R.; Webber, S. E.; Klyuyev, S.; Nudler, E.; Artsimovitch, I.; Vassylyev, D. G. Nature 2009, 457, 332−335. (9) Mariner, K.; McPhillie, M.; Trowbridge, R.; Smith, C.; O’Neill, A. J.; Fishwick, C. W. G.; Chopra, I. Antimicrob. Agents Chemother. 2011, 55, 2413−2416. (10) Schiefer, A.; Schmitz, A.; Schäberle, T. F.; Specht, S.; Lämmer, C.; Johnston, K. L.; Vassylyev, D. G.; König, G. M.; Hoerauf, A.; Pfarr, K. J. Infect. Dis. 2012, 206, 249−257.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00175. Procedures for the synthesis of substrates used in this study, along with all spectral characterization data (PDF) E
DOI: 10.1021/acs.jnatprod.5b00175 J. Nat. Prod. XXXX, XXX, XXX−XXX
