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Cystochromones, Unusual Chromone-Containing Polyketides from the Myxobacterium Cystobacter sp. MCy9104 Suvd Nadmid,†,‡ Alberto Plaza,†,‡,§ Ronald Garcia,†,‡ and Rolf Müller*,†,‡ †

Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for Infection Research (HZI) and Pharmaceutical Biotechnology, Saarland University, Campus C2 3, 66123 Saarbrücken, Germany ‡ German Center for Infection Research (DZIF), Partner site Hannover-Braunschweig 38124 Hannover-Braunschweig, Germany S Supporting Information *

ABSTRACT: Seven new chromone-containing polyketides, termed cystochromones A−G, were isolated from the myxobacterial strain Cystobacter sp. MCy9104. Their structures were elucidated using comprehensive NMR spectroscopy and HR-MS/MS. Cystochromones bear a pentadecyl moiety unusually attached at C-5 of the chromone ring. Moreover, isotope-labeled substrate feeding experiments and NMR analysis suggested a hybrid iso-fatty acid and polyketide synthase biosynthetic pathway for these secondary metabolites.

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formula of C28H42O5 requiring eight degrees of unsaturation. The 1H NMR spectrum of 1 exhibited two doublets ascribable to two meta-coupled aromatic protons at δ 6.65 (1H, d, J = 2.3 Hz, H-6) and 6.63 (1H, d, J = 2.3 Hz, H-8), an olefinic singlet at δ 6.07 (1H, s, H-3), a downfield shifted methylene singlet at δ 3.60 (2H, s, H-3′), and two singlets corresponding to three methyl groups at δ 2.26 (3H, s, H-1′) and 1.21 (6H, s, H-15″/ H-16″) (Table 1). Additionally, highly overlapped signals corresponding to methylene protons were observed at δ 1.25− 1.35 (H-3″−H-12″), indicating the presence of a saturated alkyl chain. The 13 C NMR spectrum of 1 showed signals corresponding to two carbonyls at δ 201.8 (C-2′) and 179.3 (C-4), three oxygenated aromatic quaternary carbons at δ 160.21 (C-7), 160.19 (C-9), and 159.4 (C-2), an oxygenated sp3 quaternary carbon at δ 72.0 (C-14″), and overlapped carbon resonances at δ 29−30 (C-3″−C-12″) (Table 1). Analysis of the HMBC correlations observed for the olefinic proton H-3 and the aromatic protons H-6 and H-8 established the occurrence of a 2,5,7-trisubstituted chromone ring (Figure 1). Furthermore, long-range correlations from the proton signal at δ 3.60 (H-3′) to the ketone carbonyl at δ 201.8 (C-2′) and to the methyl carbon at δ 29.7 (C-1′) revealed the presence of a propan-2-one moiety. Connectivity of the propan-2-one moiety to the chromone ring was deduced via long-range correlations from H-3′ to the aromatic carbons at δ 159.4 (C-2) and 113.7 (C-3) (Figure 1). The structure of the remaining C16H33O unit was established as a 2-methylpentadecan-2-ol residue (C-1″−C16″) on the basis of HMBC and COSY correlations. Linkage of this saturated alkyl chain to the chromone ring was secured by strong HMBC correlations from the equivalent methylene protons at δ 3.18 (H-1″) to the aromatic carbon resonances at

ver the past years, myxobacteria have attracted the attention of the scientific community due to their enormous biosynthetic capabilities of producing secondary metabolites showing novel scaffolds.1,2 The investigation of these Gram-negative bacteria has resulted in numerous new structures including the depsipeptides aetheramides,3 the unusual catecholate type of siderophores hyalachelins,4 and the macrolide glycosides disciformycins5 as well as the fascinating peptides cystobactamides, which consist of a rare para-amino benzoic acid chain.6 During our ongoing screening program aimed at the discovery of novel metabolites from terrestrial myxobacteria, Cystobacter sp. strain MCy9104 stood out due to its LC-HR-MS chromatographic profile, which showed a number of unreported metabolites. Herein, we report the isolation, structure elucidation, and first insights into the biosynthesis of a new group of chromone-containing natural products, named cystochromones A−G (1−7). Their structures were elucidated using a combination of HR-MS/MS and 2D NMR experiments. Moreover, a biosynthetic scheme was proposed on the basis of results from feeding experiments employing isotopically labeled precursors.

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RESULTS AND DISCUSSION The myxobacterial strain MCy9104 was cultivated in CLF production medium in the presence of 2.5% v/v of the neutral adsorber resin Amberlite XAD-16. After 10 days the resin was harvested by sieving, lyophilized, defatted with hexane, and subsequently extracted with ethyl acetate. The resultant dark brown, oily, crude extract was fractionated by size exclusion chromatography and further subjected to semipreparative HPLC, yielding seven new chromone derivatives, termed cystochromones A−G (1−7). HR-ESIMS of cystochromone A (1) gave a quasimolecular ion at m/z 459.3103 [M + H]+ corresponding to a molecular © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 21, 2015

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DOI: 10.1021/acs.jnatprod.5b00343 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. NMR Spectroscopic Data for Cystochromones A (1) and D (4) 1a pos 2 3 4 5 6 7 8 9 10 1′ 2′ 3′a 3′b 1″ 2″ 3″-4″ 5″ 6″ 7″ 8″ 9″a 9″b 10″−12″ 13″ 14″ 15″/16″

δC, type c

159.4, C 113.7, CH 179.3, C 148.4, C 116.5, CH 160.21, C 101.5, CH 160.19, C 115.3, C 29.7, CH3 201.8, C 48.7, CH2 35.3, CH2 31.3, CH2 29.2−30.1, 29.2−30.1, 29.2−30.1, 29.2−30.1, 29.2−30.1, 29.2−30.1,

4b δH (J in Hz) d

3.60, s

166.5, 112.4, 181.3, 148.2, 117.6, 163.4, 101.7, 161.5, 114.8, 23.4, 66.1, 44.0,

3.18, t (7.5) 1.56, m 1.25−1.35, m 1.25−1.35, m 1.25−1.35, m 1.25−1.35, m 1.25−1.35, m 1.25−1.35, m

36.1, CH2 32.4, CH2 30.3−30.7, CH2 24.2, CH2 38.5, CH2 215.2, C 77.9, CH 34.5, CH2

1.25−1.35, m 1.46, t (6.7)

30.3−30.7, CH2 44.5, CH2 71.2, C 29.0, CH3

6.07, s

6.65, d (2.3) 6.63, d (2.3)

2.26, s

CH2 CH2 CH2 CH2 CH2 CH2

29.2−30.1, CH2 44.1, CH2 72.0, C 29.3, CH3

δC, type e

1.21, s

C CH C C CH C CH C C CH3 CH CH2

δHf (J in Hz) 6.06, s

6.64, d (2.3) 6.67, d (2.3)

1.28, d (6.2) 4.19, m 2.66, dd (7.9, 14.4) 2.72, dd (4.9, 14.4) 3.16, t (7.5) 1.57, m 1.31−1.40, m 1.58, m 2.55, m 4.06, q (3.8) 1.53, m 1.72, m 1.35−1.40, m 1.44, t (6.7) 1.16, s

Recorded in CDCl3. bRecorded in MeOD4. cAcquired at 125 MHz, referenced to solvent signal CDCl3 at δ 77.23 ppm. dAcquired at 500 MHz, referenced to solvent signal CDCl3 at δ 7.24 ppm. eAcquired at 175 MHz, referenced to solvent signal MeOD4 at δ 49.15 ppm. fAcquired at 700 MHz, referenced to solvent signal MeOD4 at δ 3.31 ppm. a

δ 148.4 (C-5), 116.5 (C-6), and 115.3 (C-10) (Figure 1). The planar structure of 1 was established as a chromone ring bearing an alkyl chain in position C-5 and a propane-2-one moiety at C-2. HR-ESIMS supported the molecular formulas C28H42O4 and C28H42O6 for cystochromones B (2) and C (3), respectively. Detailed analysis of 2D NMR data of 2 and 3 in comparison to that of 1 clearly indicated that cystochromones B and C were

Figure 1. Key HMBC (arrow) and COSY (bold line) correlations of 1.

B



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the 14″-deoxy and 15″-hydroxy derivatives of 1, respectively (Table S2). Cystochromone D (4) showed a quasimolecular ion peak at m/z 491.3001 [M + H]+ ascribable to a molecular formula of C28H42O7. The HSQC spectrum of 4 closely resembled that of 1, differing only by the existence of two additional oxymethine signals (δH‑2′ 4.19, δC‑2′ 66.1 and δH‑8″ 4.06, δC‑8″ 77.9) (Table 1). Moreover, COSY correlations observed between protons H1′/H-2′/H-3′ established the presence of a propan-2-ol moiety (Figure 2). In turn, HMBC correlations from methylene

Figure 3. Key HMBC (arrows) and TOCSY/COSY (bold lines) correlations of 6.

moiety to C-7 of the chromone ring, thereby completing the planar structure of 6 as depicted in Figure 3. The absolute configuration of α-rhamnose was determined to be L by GC-MS analysis of a trimethylsilylated (+)-2-butyl derivative of hydrolyzed 6 in comparison to the trimethylsilylated (−)-2- and (+)-2-butyl derivative of an authentic L-αrhamnose standard (Figure S3).7 Cystochromone G (7) displayed an ion peak at m/z 445.2965, which was 14 mass units lower than that of 1. In addition, its NMR data showed high similarity to those of 1, indicating 7 to differ from 1 only by having one methylene unit less in the saturated alkyl chain (Table S2). In order to determine the absolute configuration of C-2′ and C-8″ in 4 by applying Mosher’s method,8 we first prepared its 7-O-methyl derivative 4a (Figure 2) by treatment with iodomethane and sodium methoxide. Subsequent esterification of 4a (0.3 mg) was performed with R-(+)- and S-(−)-MTPA acid to produce the respective triesters. MS analysis of both derivatives showed a formal elimination of one MTPA-ester residue. NMR spectra corroborated the elimination from the C2′/C-3′ position, which results in a trans-Δ2′ unsaturation (Table S6). Furthermore, 1H NMR and HSQC spectra of both di-MTPA esters (S and R) exhibited two sets of signals each. This led us to speculate that keto−enol tautomerization might have occurred during the esterification reaction, inducing epimerization at C-8″, and therefore we were unable to establish the configuration of C-8″. Biosynthesis of Cystochromones. The biosynthetic origin of cystochromones was studied by feeding experiments using 13C-labeled sodium acetate and [D10]leucine. The 13C NMR spectrum of 1 isotope labeled with [1-13C]acetate clearly showed enhanced signal intensity of alternate carbons starting from the C-2′ keto carbonyl, including chromone carbons C-2, C-4, C-5, C-7, and C-9 and part of the alkyl carbons C-1″ to C10″ (Scheme 1). Similarly, incorporation of [2-13C]acetate was observed for the remaining carbons in the chromone ring, C11″, C-1′, and C-3′. This result was in agreement with the expected polyketide origin of 1. Analysis of 13C−13C coupling constants resulting from a [13C2]acetate feeding experiment allowed us to establish the incorporation of intact acetate units (Scheme 1 and Table 2). Surprisingly, the carbon resonance of C-1′ was observed as a singlet, although it was enriched by feeding the producing cultures with [2-13C]acetate. Altogether, these data suggested a decarboxylation from the polyketide chain. Moreover, the C12″−C-16″ portion of the molecule was not labeled with acetate carbons, suggesting that its origin could be derived from leucine via isovaleryl-CoA being used as starter molecule. Indeed, LC-MS analysis of an extract prepared from a culture fed with L-[D10]leucine revealed a mass shift of 8 Da for compounds 4 and 6, presenting intact incorporation of

Figure 2. Key COSY (bold line) and HMBC (arrow) correlations of 4.

protons H-3′ to the carbons C-2 and C-3, together with HMBC correlations from H-2′ to C-2, linked the propanol moiety to C-2 of the chromone ring (Figure 2). HMBC and COSY correlations and the MS2 fragmentation experiment allowed us to identify the alkyl chain of 4 as 8,14dihydroxy-14-methylpentadecan-7-one. In particular, HMBC correlations from the methylene protons H-9″ (δ 1.53, 1.72), H-6″ (δ 2.55), and H-5″ (δ 1.58) and from the oxymethine proton H-8″ (δ 4.06) to the ketone carbon resonance at δ 215.2 (C-7″) determined the presence of an α-ketol functionality (Figure 2). The exact position of this functionality was proposed by an ESIMS2 fragmentation experiment. Indeed, the ESIMS2 spectrum of the quasimolecular ion peak at m/z 491 [M + H]+ gave a daughter ion peak at m/z 455 [M + H − 36]+ corresponding to loss of two water molecules. Additionally, a daughter ion peak at m/z 331 [M + H − 160]+ was observed, which suggested an α-cleavage at C-7″ (Figure S1). Taken together, MS2 and NMR data indicated that the keto group should be placed at C-7″ and the hydroxy group at C-8″. Finally, linkage of the 8,14-dihydroxy-14-methylpentadecan-7one residue at C-5 of the chromone ring was deduced through the strong HMBC correlations from the proton at δ 3.16 (H1″) to the aromatic carbons δ 148.2 (C-5), 117.6 (C-6), and 114.8 (C-10). Cystochromone E (5) showed a quasimolecular ion peak at m/z 493.3164 [M + H]+ corresponding to a molecular formula of C28H44O7, which was two mass units higher than 4. 2D NMR data of 5 were almost identical to those of 4, except for the substitution of a hydroxy group at C-7″ in 5 for the keto group in 4 (Table S4). HR-ESIMS at m/z 621.3650 [M + H]+ of cystochromone F (6) suggested a molecular formula of C34H52O10. HSQC, TOCSY, and COSY data of 6 indicated the presence of an αrhamnose moiety (Table S5). Moreover, 2D NMR data of the aglycone unit of 6 were almost identical to those of 4, only differing by the resonances belonging to the alkyl chain. Sequential COSY correlations from H-1″ to H-5″ along with HMBC correlations from H-5″ and the proton signals at δ 1.60 (H-4″), 2.44 (H-7″), and 1.55 (H-8″) to the keto resonance at δ 214.3 (C-6″) clearly determined the alkyl chain of 6 to be 14hydroxy-14-methylpentadecan-6-one (Figure 3). An HMBC correlation from the anomeric proton at δ 5.57 (H-1″′) to the carbon resonance at δ 160.6 (C-7) attached the rhamnose C



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Scheme 1. Proposed Biogenetic Pathway for 1 Deduced from Feeding Studies

Table 2. 13C NMR Data Indicating the Incorporation of [1,2-13C]Acetate into Cystochromone A (1) carbon 2 3 4 5 6 7 8 9 10 a

δC 159.4 113.7 179.3 148.4 116.5 160.21 101.5 160.19 115.3

JC−C (Hz)

correlated carbon

71.2 71.3 57.0 41.3 63.1 63.4 73.0 73.0 56.7

carbon

C-3 C-2 C-10 C-1″ C-7 C-6 C-9 C-8 C-4

1′ 2′ 3′ 1″ 2″ 3″-12″ 13″ 14″ 15″/16″

b

c

δ Ca

JC−C (Hz)

29.7 201.8 48.7 35.3 31.3 29.2−30.1 44.1 72.0 29.3

b

37.0 37.6 41.1

correlated carbon C-3′ C-2′ C-5

c c d d d

d

Referenced to solvent signal CDCl3 at 77.23 ppm. Enriched, but appeared as a singlet. Overlapped signals. Not enriched with 13C-acetates.

that the cystochromones may be formed by a hybrid iso-fatty acid and polyketide synthase biosynthetic pathway.

isovalerate (Figure S4). It is worth mentioning that the shift of 8 Da is in concordance with loss of two deuterium atoms due to the transamination of leucine to [D9]-α-ketoisocaproic acid9 (Scheme S1) and oxidation at C-14″. These results indicate that cystochromones are assembled from a leucine-derived fatty acid (FA),10 which serves as a starter unit for the respective polyketide synthase (PKS). On the basis of our feeding experiments, we postulate that the presumed hybrid biosynthetic pathway of 1 initiates with the formation of leucine-derived isovaleryl-CoA, which further undergoes branched chain FA biosynthesis to yield a 15methylhexadecanoic acid (1a)10 (Scheme 1 and S1), which is known to be one of the predominant FAs in myxobacteria.11 We suggest that after activation the extension of this FA with six malonyl-CoA units is carried out using standard polyketide assembly. However, we cannot rule out the possibility of one very large polyketide synthase generating the complete polyketide chain. Independent of this question, the resulting polyketide undergoes cyclization and aromatization steps to form a characteristic chromone moiety, followed by post-PKS modification, i.e., decarboxylation, regiospecific hydroxylation, and glycosylation to form 5. Cystochromone A (1) was tested for activities against both Gram-negative and Gram-positive bacteria, cancer cell lines, and various fungal strains. It did not show significant inhibitory activity up to concentrations as high as 64 μg/mL. To the best of our knowledge, this is the first report of a chromone-containing secondary metabolite with a pentadecyl substitution at C-5.12 Chromones, including stigmatellin X, a chromone derivative also isolated from myxobacteria,13 usually bear an alkyl chain in position 2. Stigmatellins are assembled by a polyketide synthase using acetate and propionate building blocks, while aromatization occurs during or after the polyketide chain generation.14 Our feeding experiments show

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EXPERIMENTAL SECTION

General Experimental Procedures. The specific optical rotations were measured on a JASCO P-2000 polarimeter. UV spectra were measured on a Thermo Scientific Nanodrop 2000c spectrophotometer, and IR spectra were obtained on a PerkinElmer FT-IR Spectrum One spectrometer. NMR spectra were acquired on Bruker Ascend 500 and 700 MHz NMR spectrometers equipped with a 5 mm TXI cryoprobe. Deuterated MeOD4 and CDCl3 were used as NMR solvents, and HSQC, HMBC, 1H−1H COSY, 2D TOCSY, and ROESY spectra were recorded using standard pulse programs. LCHRMS data were obtained on a maXis 4G mass spectrometer (Bruker Daltonics, Germany) coupled with a Dionex Ultimate 3000 RSLC system, using a BEH C18, 100 × 2.1 mm, 1.7 μm dP column (Waters, Germany) with a linear gradient of 5−95% ACN + 0.1% FA in H2O + 0.1% FA at 600 μL/min in 18 min with UV detection in the 200−600 nm range. Mass spectra were acquired using the ESI source in the range from 150 to 2000 m/z at 2 Hz scan speed. Strain Isolation and Identification. The strain MCy9104 was isolated in 2006 from a soil sample collected in Manila, Philippines. The bacterium was discovered by a standard myxobacterial baiting method using rabbit dung pellets, which were partly depressed in the soil.15,16 It was recognized as a myxobacterium by the development of rounded and brownish fruiting bodies on the baits. By picking them up using a sterile fine needle and inoculating onto buffered water agar with Escherichia coli spots (20 μL) and for successive transfers of the farthest swarm colony edge,17 the strain was finally purified. The isolate was maintained in buffered VY/2 agar and permanently stored in a −80 °C freezer using 50% glycerol as a cryoprotectant. Based on the complete (1524 bp) 16S rRNA gene sequence (GenBank accession number KT070190), strain MCy9104 shows closest similarity (99%) with Cystobacter f uscus DSM 2262T (GenBank accession number NR_043941), Cystobacter ferrugineus Cb fe18T (NR_025343), and Cystobacter badius DSM14723T (NR_043940). D



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sieving, lyophilized, defatted with n-hexane, and extracted with 3 × 300 mL of ethyl acetate. EtOAc extracts were then subjected to Agilent semipreparative HPLC equipped with an automatic fraction collector and a Luna C18(2), 250 × 10 mm, 5 μm dP column. Fractionation was carried out using a linear gradient started with 10% B (ACN + 0.1% FA) and increased to 50% in 15 min and to 100% B in 30 min. Fractions of interest were combined and concentrated under reduced pressure, and final purification was achieved on the semipreparative HPLC system equipped with a Synergi Fusion 250 × 10 mm, 4 μm dP column with a linear gradient of 75−100% B in 20 min at 2.5 mL/min flow rate to yield enriched 1 in 1.0 mg ([1-13C]-acetate), 1.2 mg ([2-13C]-acetate), and 0.3 mg ([1,2-13C]-acetate). The feeding experiment with labeled leucine was achieved with 0.1 mM L-[D10]leucine (in 2 mL of DMSO), which was added as two equal portions to the culture at 18 and 24 h after cultivation initiation. The MeOH extract of XAD was analyzed by LC-HR-MS. Methylation and Preparation of (R)- and (S)-MTPA Esters of 4. Starting material 4 (1 mg) was mixed in methyl iodide (100 μL) with 0.5 M sodium methoxide solution in methanol (30 μL) at room temperature. After 2, 24, and 29 h another three portions of both reagents were added. The mixture was dried under a N2 stream after 46 h and resuspended in MeOH. The methylated product 4a was purified by semipreparative HPLC equipped with a Luna C18(2) 250 × 10 mm, 5 μm dP column with a linear gradient of 10−100% B (ACN + 0.1% FA) in 25 min. To the solution of 4a (0.35 mg in 100 μL of dried chloroform) were added (R)-(+)- and (S)-(−)-α-methoxy-α-trifluoromethyl phenylacetic acid (MTPA, 150 μL), extradry pyridine (20 μL), N,N′diisopropylcarbodiimide (DIC, 140 μL), and an excess amount of dimethylaminopyridine in four portions at 0, 24, 48, and 72 h. The reactions were monitored by LC-MS. After 4 days, the reaction mixture was dried under N2, dissolved in water, and extracted with ethyl acetate. The organic layer was concentrated and analyzed by LCMS. Purification of the (R)- and (S)-Mosher ester of 4a was carried out by semipreparative HPLC equipped with a Luna C18(2) 250 × 10 mm, 5 μm dP column under a linear gradient of 10−65% B in 15 min, increased up to 100% in 30 min, followed by an isocratic run at 100% for another 9 min, with a 2.5 mL/min flow rate. Mosher esters were eluted at 56 min. Assigment of Absolute Configuration of Rhamnose. Approximately 0.3 mg of 6 was hydrolyzed with 1 N HCl (0.25 mL) at 60 °C for 4 h. After cooling to room temperature, the solution was concentrated by a Speedvac concentrator (Eppendorf). The residue was dissolved in (+)-2-butanol (0.3 mL) and acetyl chloride (5 μL) and stirred for 8 h at 80 °C. After cooling, the reaction was quenched with saturated aqueous NaHCO3 (0.5 mL) and subsequently extracted with ethyl acetate (2 × 0.5 mL). The organic layer was dried first with a stream of N2, followed by overnight drying in vacuo, and then treated with 1-(trimethylsilyl)imidazole (40 μL) and pyridine (160 μL) for 15 min at 60 °C. The solution was dried using a Speedvac concentrator, and the residue was separated by H2O and EtOAc (2 × 400 μL, 1:1 v/v). The organic layer was analyzed by GCMS on an Agilent 6890N gas chromatograph with a 5973 electron impact mass selective detector and a 7683B injector (Agilent) using a dimethyl-(5% phenyl)-polysiloxane capillary column (Agilent DB-5ht, 0.25 mm by 30 m by 0.1 μm). Helium was used as the carrier gas at a flow rate of 1 mL/min. A 5 μL portion of the sample was injected in split mode (split ratio, 10:1). The column temperature was increased from 50 °C to 180 °C at a rate of 5 °C/min. Other temperatures were as follows: inlet, 275 °C; GC-MS transfer line, 280 °C; ion source, 230 °C; and quadrupole, 150 °C. The mass selective detector was operated in scan mode, scanning the mass range from m/z 40 to 700. Peaks corresponding to the trimethylsilylated (+)-2-butyl rhamnoside were coeluted with the same derivative of authentic L-rhamnose (eluted at 22.26 min) that was treated in the same manner. Since the retention times of (+)-2-butyl L-rhamnoside and (−)-2-butyl D-rhamnoside are the same, the authentic L standard was derivatized with (−)-2-butanol and analyzed by GC-MS (eluted at 22.07 min).

The strain’s affiliation to the genus Cystobacter was also supported by morphological characteristics and phylogenetic analysis (Figure S2). Strain Cultivation. Preculture of strain MCy9104 Cystobacter sp. grew in CLF medium (4 g of fructose, 6 g of glucose, 10 g of skim milk, 2 g of yeast extract, 1 g of CaCl2 × 2H2O, 1 g of MgSO4 × 7H2O, 11.9 g of HEPES per liter, pH 7.0 adjusted with 10 N KOH) on a rotary shaker at 30 °C. Four-day-old preculture was inoculated (2% v/ v) into 12 L of CLF production medium (distributed in 6 × 5 L shaking flasks) and placed on a rotary shaker at 180 rpm in a 30 °C room, and adsorber resin Amberlite XAD-16 (2.5% v/v) was added on day 9. XAD was collected on the following day by sieving and lyophilized overnight. Extraction and Isolation. Dry XAD was defatted with n-hexane and extracted with ethyl acetate 3 × 500 mL. One gram of dark brown, oily, crude extract was obtained after evaporation of ethyl acetate in vacuo, and the crude extract was then subjected to Sephadex-LH 20 column chromatography with MeOH as eluent. The fractions containing cystochromones were dried using a Genevac and purified by a semipreparative HPLC Agilent system equipped with a Luna C18(2), 250 × 10 mm, 5 μm dP column. The linear gradient started with 10% B (ACN + 0.1% FA) for 5 min, increased to 50% in 15 min, and to 100% in 30 min at 2.5 mL/min, followed by an isocratic run for 10 min at 3 mL/min to afford compounds 1 (1 mg, tR = 39.1 min), 2 (0.2 mg, tR = 53.7 min), 3 (0.4 mg, tR = 31.0 min), 4 (0.3 mg, tR = 20.5 min), 5 (0.3 mg, tR = 19.8 min), 6 (0.7 mg, tR = 21.6 min), and 7 (0.5 mg, tR = 37.2 min). Cystochromone A (1): colorless solid; [α]20D −12.9 (c 0.2 in MeOH); IR (film) νmax 3681, 2938, 2865, 1455, 1346, 1055, 1032 cm−1; UV(MeOH) λmax (log ε) 227 (4.27), 251 (4.21), 294 (4.06) nm; 1H and 13C NMR data see Tables 1 and S1; HR-ESIMS m/z 459.3103 [M + H]+ (calcd for C28H43O5 459.3105). Cystochromone B (2): colorless solid; [α]20D −22.5 (c 0.15 in MeOH/CH2Cl2, 1:1); IR (film) νmax 2920, 2851, 1652, 1611, 1352 cm−1; UV(MeOH) λmax (log ε) 220 (4.56), 252 (4.41), 292 (4.28) nm; 1H and 13C NMR data see Table S2; HR-ESIMS m/z 443.3150 [M + H]+ (calcd for C28H43O4 443.3155). Cystochromone C (3): colorless solid; [α]20D −11.6 (c 0.2 in MeOH); IR (film) νmax 3372, 2923, 2853, 1647, 1600 cm−1; UV(MeOH) λmax (log ε) 218 (4.11), 251 (3.92), 295 (3.81) nm; 1 H and 13C NMR data see Table S2; HR-ESIMS m/z 475.3051 [M + H]+ (calcd for C28H43O6 475.3054). Cystochromone D (4): colorless solid; [α]20D −4.7 (c 0.2 in MeOH); IR (film) νmax 3328, 2932, 2857, 1643, 1598, 1390 cm−1; UV(MeOH) λmax (log ε) 219 (3.59), 251 (3.42), 290 (3.27) nm; 1H and 13C NMR data see Tables 1 and S3; HR-ESIMS m/z 491.3001 [M + H]+ (calcd for C28H43O7 491.3003). Cystochromone E (5): colorless solid; [α]20D −8.0 (c 0.2 in MeOH); IR (film) νmax 3708, 2923, 2866, 1738, 1455, 1365, 1228, 1055 cm−1; UV(MeOH) λmax (log ε) 218 (4.15), 252 (4.03), 293 (3.89) nm; 1H and 13C NMR data see Table S4; HR-ESIMS m/z 493.3164 [M + H]+ (calcd for C28H45O7 493.3159). Cystochromone F (6): colorless solid; [α]20D −28.0 (c 0.2 in MeOH); IR (film) νmax 3352, 2926, 2851, 1643, 1598, 1395 cm−1; UV(MeOH) λmax (log ε) 228 (4.29), 251 (4.09), 280 (4.00) nm; 1H and 13C NMR data see Table S5; HR-ESIMS m/z 621.3650 [M + H]+ (calcd for C34H53O10 621.3633). Cystochromone G (7): colorless solid; [α]20D −20.8 (c 0.1 in MeOH/CH2Cl2, 1:1); IR (film) νmax 2929, 2851, 1646, 1598 cm−1; UV(MeOH) λmax (log ε) 217 (3.76), 248 (3.63), 294 (3.54) nm; 1H and 13C NMR data see Table S2; HR-ESIMS m/z 445.2965 [M + H]+ (calcd for C27H41O5 445.2954). Stable Isotope Feeding. Feeding experiments were carried out using [1-13C]-, [2-13C]-, and [1,2-13C2]-acetate, each in a 5 mM final concentration in 2 L cultures. Precursors were dissolved in a mixture of 7 mL of sterile water and 2 mL of DMSO and added into the culture as three equal portions at 20, 28, and 44 h after inoculation. Simultaneously, positive and negative control cultures were prepared with 5 mM unlabeled sodium acetate and 2 mL of DMSO, respectively. After 5 days of inoculation, 50 mL of XAD-16 was added and harvested after another 2 days. XAD was collected by E



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(13) Kunze, B.; Kemmer, T.; Höfle, G.; Reichenbach, H. J. Antibiot. 1984, 37, 454−461. (14) Gaitatzis, N.; Silakowski, B.; Kunze, B.; Nordsiek, G.; Blöcker, H.; Höfle, G.; Müller, R. J. Biol. Chem. 2002, 277, 13082−13090. (15) Krzemieniewska, H.; Krzemieniewski, S. Myxobacteria of the Species Chondromyces Bull. Acad. Polon. Sci. Lett. Classe Sci. Math Nat. Ser. B; Berkeley and Curtis, 1946; Vol. 1, pp 31−48. (16) Shimkets, L.; Dworkin, M.; Reichenbach, H. The myxobacteria. In The Prokaryotes, 3rd ed.; Dworkin, M.; Falkow, S.; Rosenberg, E.; Schleifer, K.-H.; Stackebrandt, E., Eds.; Springer-Verlag: Berlin, 2006; Vol. 7, pp 31−11. (17) Garcia, R.; Müller, R. Family Polyangiaceae. In The Prokaryotes; Rosenberg, E.; DeLong, E.; Lory, S.; Stackebrandt, E.; Thompson, F., Eds.; Springer-Verlag: Berlin, 2014; pp 247−279.

ASSOCIATED CONTENT

S Supporting Information *

Tabulated 1H and 13C NMR assignment for 1−7, 1D and 2D NMR spectra for 1−7, 13C NMR spectra of acetate-enriched 1, ESIMS2 fragmentation of 4, GC-MS spectra of derivatized 4, LC-MS data and calculation of 4 and 6 for [D10]leucine fed experiment, NMR assignment of di(S)-Mosher ester of 4a, and phylogenetic tree based on 16S rRNA of the strain MCy9104. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00343.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +49 681 30270201. Fax: +49 681 30270202. E-mail: rolf. [email protected]. Present Address §

Fraunhofer IME, Sanofi-Fraunhofer Natural Products Center of Excellence, Industriepark Hö chst, Bldg. G878, 65926 Frankfurt/Main, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.N. is grateful to the Deutscher Akademischer Austauschdienst (DAAD) and Helmholtz Centre for Infection Research (HZI) for Ph.D. scholarships. We thank our colleagues T. Hoffmann and J. Hermann (HIPS) for performing the MS/MS fragmentation and bioactivity evaluation tests, respectively. We would like to acknowledge K. Gemperlein (HIPS) for help with absolute configuration assignment of rhamnose and GC measurements. We also express our gratitude to K. Harmrolfs and J. Hoffmann (HIPS) for advice and discussion on derivatization reactions and NMR data interpretation of Mosher esters as well as proofreading of the manuscript. Research in R.M.’s laboratory was funded by the Bundesministerium für Bildung und Forschung and Deutsche Forschungsgemeinschaft.

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REFERENCES

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