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Indiacens A and B: Prenyl Indoles from the Myxobacterium Sandaracinus amylolyticus Heinrich Steinmetz,† Kathrin I. Mohr,† Wiebke Zander,† Rolf Jansen,† Klaus Gerth,† and Rolf Müller*,†,‡ †
Research Group Microbial Drugs, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany Helmholtz Institute Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Saarland University, Postfach 151150, 66041 Saarbrücken, Germany
‡
S Supporting Information *
ABSTRACT: The gliding bacterium Sandaracinus amylolyticus, strain NOSO-4T, was recently characterized as the first representative of a new myxobacterial genus. A screening of the culture broth for antibiotically active metabolites followed by isolation and characterization revealed two unique 3formylindol derivatives, indiacen A (1) and its chloro derivative indiacen B (2). Both are active against Grampositive and Gram-negative bacteria as well as the fungus Mucor hiemalis. The biosynthetic origin of the isoprene-like side chain in 1 and 2 was studied by in vivo feeding experiments with 13C-labeled precursors.
D
uring the last three decades myxobacteria became highly respected for their ability to produce structurally diverse secondary metabolites.1,2 More than 100 new basic structures with about 500 variants exhibiting numerous biological activities substantiate the high biosynthetic potential of myxobacteria.3,4 Throughout our screening program for biologically active natural products, new species of myxobacteria were the most promising sources of new metabolites. The recently characterized gliding bacterium Sandaracinus amylolyticus is the first representative of a novel family of myxobacteria,5 and herein we report the isolation, structure elucidation, biosynthesis, and antimicrobial activities of two secondary metabolites, indiacen A (1) and the chlorinederivative indiacen B (2) from this organism. The culture broth of S. amylolyticus strain NOSO-4T was screened for the presence of new antimicrobial secondary metabolites using antimicrobial bioassays coupled with HPLCUV-HRESIMS analysis and comparison with our in-house database. Subsequent investigations of this culture led to the discovery of two new 3-formylindol derivatives, indicen A (1) and its chlorine derivative indicen B (2) (Figure 1).
HRESIMS of indiacen A (1) indicated the molecular formula C14H14NO. A strong band at 1641 cm−1 in the IR spectrum suggested the presence of a carbonyl group. The complete structure was elucidated by 1D and 2D NMR spectroscopy (Table 1 and Supporting Information). Analysis of the HMQC spectrum showed the presence of four aromatic and three olefinic methines together with one vinylic methyl group (δH 2.14, δC 19.4). Additionally, the HMQC spectrum indicated the presence of an aldehyde group (δH 9.92, δC 184.4), corroborating the results of IR analysis. HMBC and COSY correlations indicated a 3,4-disubstituted indole system (Figure 2). HMBC correlations between the aldehyde proton H-13 and the indole carbons C-2, C-3, and C-3a (δC 142.7, 121.8, and 124.1) and between the H-2 singlet at δH 8.28 and the carbonyl C-13 at δC 184.4 established the presence of a 3-formylindole system. Additionally, long-range correlations from the vinylic methyl group at C-12 to the olefinic carbons C-9, C-10, and C11 (δ 132.2, 144.5, and 116.5) along with a COSY correlation between both olefinic protons H-8 (δ 8.47) and H-9 (δ 6.96) constituted the 3-methyl-1,3-butadienyl moiety. The connection of this moiety to the indole ring was obtained from HMBC correlations of the proton signal at H-8 (δ 8.47) to indole carbons C-4, C-5, and C-3a (δC 133.1, 119.1, and 124.1), linking the butadienyl chain to the indole ring at C-4. The geometry of the Δ8,9 double bond was assigned as trans on the basis of the large coupling constant 3J8,9 of 16.3 Hz and ROESY correlations between H-8 and Me-12. The HRESIMS of indiacen B (2) displayed an isotopic pattern of the molecular ion cluster [M + H]+, suggesting the
Figure 1. Structures of indiacens A (1) and B (2).
Received: April 28, 2012 Published: October 4, 2012
© 2012 American Chemical Society and American Society of Pharmacognosy
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Table 2. Minimum Inhibitory Concentration (MIC) in μg/ mL of Indiacens A (1) and B (2)
Table 1. NMR Spectroscopic Data of Indiacens A (1) and B (2) (acetone-d6, 1H 400 MHz, 13C 100 MHz)a 1 position
δC, mult
2 3 3a 7a 7 6
142.7, 121.8, 124.1, 140.1, 112.3, 125.0,
CH C C C CH CH
5 4 8 9 10 11a 11b 12 13
119.1, 133.1, 131.7, 132.2, 144.5, 116.5,
CH C CH CH C CH2
19.4, CH3 184.4, CH
2 δH, mult (J in Hz)
8.28, s
7.44. d (8.1) 7.28, dd (8.1, 7.6) 7.59, d (7.6) 8.47, d (16.3) 6.96, d (16.3) 5.12, 5.05, 2.14, 9.92,
s s s s
δC, mult 143.0, 121.7, 124.1, 140.2, 112.5, 125.1,
CH C C C CH CH
119.0, 132.7, 131.8, 128.8, 139.6, 119.4,
CH C CH CH C CH
13.4, CH3 184.5, C
δH, mult (J in Hz) 8.30, s
7.46, d (8.1) 7.28, dd (8.1, 7.7) 7.58, d (7.7)
a
test organisms
1
2
Arthrobacter rubellus Nocardioides simplex Mycobacterium lacticola Mycobacterium diernhoferi Pseudomonas stutzeri Chromobacterium violaceum E. coli TOL Ca Mucor hiemalis
16.6 8.3 16.6 16.6 >33.0 16.6 16.6 16.6
0.8 3.3 33.0 >33.0 33.0 33.0 33.0 16.6
Mutant strain with increased cell wall permeability.
Indiacens A (1) and B (2) are the first reported secondary metabolites from S. amylolyticus. Previously, 1 has been known as a synthetic precursor in the synthesis of the ergot alkaloid (±)-6,7-secoagroclavine8 but not as a natural product. Other natural dehydroprenylindole derivatives bearing a 3-methyl-1,3butadienyl side chain have been isolated from seeds of Monodora tenuifolia,9 and an indole-carboxylic acid was isolated from an unidentified fungus and described to function as a transcriptional regulator. Featuring a unique chlorinated isoprene-like side chain, the structural variant indiacen B (2) represents another chlorine metabolite from myxobacteria besides the previously reported gem-dichloro-1,3-dione chlorotonil from Sorangium cellulosum11 and chondrochloren12 from Chondromyces crocatus.
8.62, d (16.1) 6.96, d (16.1) 6.46, s 2.20, s 9.91, s
a ROESY correlations of 1: 5-H/9-H, 8-H/12-H, 9-H/11-Ha, 11-Hb/ 12H, 2-H/13-H; for 2: 5-H/9-H, 8-H/12-H, 9-H/11-H, 2-H/13-H.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were determined with a Perkin-Elmer 241 instrument, UV spectra were recorded with a Shimadzu UV-2450 UV−vis spectrophotometer, and IR spectra were measured with a Nicolet 20DXB FT-IR spectrometer. NMR spectra were recorded on a Bruker ARX 400 (1H 400 MHz, 13C 100 MHz) spectrometer. HRESIMS mass spectra were obtained with an ESI-TOF-MS (Maxis, Bruker) attached to an Agilent 1200 series UHPLC-UV [column 2.1 × 50 mm, 1.7 μm, C18 Acquity UPLC BEH (Waters), solvent A: H2O + 0.1% formic acid; solvent B: ACN + 0.1% formic acid, gradient: 5% B for 0.5 min increasing to 100% B in 19.5 min, maintaining 100% B for 5 min, flow rate 0.6 mL/min, UV detection 200−500 nm]. Cultivation of Sandaracinus amylolyticus (NOSO-4T). Sandaracinus amylolyticus (NOSO-4T) was isolated at the HZI (former GBF) in 1996 from a soil sample that had been collected in 1995 in Lucknow, Uttar Pradesh, India, using the methods of Reichenbach and Dworkin.13 The strain had been stored at −80 °C. The strain was transferred to 1 L of liquid medium consisting of 0.2% defatted soy flour, 0.2% glucose, 0.8% starch (Cerestar), 0.2% yeast extract, 0.1% CaCl2·2H2O, 0.1% MgSO4·7H2O, 50 mM HEPES, and 8 mg/L FeEDTA at pH 7.4. The preculture was supplemented with 10 mL of vitamin solution.14 Ten liters of the same medium supplemented with 1% Amberlite XAD-16 resin (Rohm & Haas) was inoculated with the preculture and incubated at 30 °C for 14 days (blade impeller 100 rpm, aeration 0.1 vvm, 20% pO2 were regulated). The XAD resin was harvested from the culture broth by sieving. Isolation of Indiacens A (1) and B (2). The XAD resin was eluted with acetone (800 mL) overnight and evaporated to an aqueous mixture. This was diluted with water and extracted with ethyl acetate (three portions of 200 mL). The combined organic layers were extracted with water and dried over Na2SO4. The ethyl acetate layer was evaporated and gave 593 mg of crude extract. The extract was dissolved in methanol (4.5 mL) and DMSO (0.5 mL) and separated by RP-MPLC chromatography [column 480 × 30 mm (Kronlab), ODS-AQ C18 (YMC), 120 Å, S 16 μm; solvent A: H2O−methanol 6/ 4; solvent B: methanol; gradient: 0% B to 50% B in 60 min, 50% B for
Figure 2. COSY and HMBC correlations of indiacen A (1).
chlorine-containing molecular formula of C14H13ClNO. 1H NMR spectra and 2D NMR correlations of 2 were almost identical to those of 1 (Table 1 and Supporting Information), with the exception of the signals corresponding to the exomethylene group. In particular, only one of the exomethylene signals was detected at δH 6.46, significantly shifted downfield compared to indiacen A (δH‑11 5.12). Thus, 2 was identified as the C-11 chloro derivative of 1. A strong ROESY correlation between H-11 and H-9 (δH 6.96) and between H-8 (δH 8.62) and Me-12 (δH 2.20) led to the assignment of the Econfiguration of the Δ10,11 double bond. The biosynthetic origin of the prenyl side chain of indiacens A (1) and B (2) was studied by feeding experiments using [1-13C]acetate, [1,2-13C2]acetate, L-[methyl-13C]methionine, [2-13C]propionate, and [1,2-13C2]mevalonolactone. When cultures of S. amylolyticus were fed with [1,2 13 C 2 ]mevalonolactone, the subsequently isolated indiacens A (1) and B (2) were significantly 13C-enriched (66% and 42% at C11, respectively), whereas all other feeding experiments failed to show significant incorporations. While the 3-methyl-1,3butadienyl side chain originates from mevalonolactone (or mevalonate), the indole moiety can be expected to result from tryptophan, as has been shown for various other indole derivatives from myxobacteria.7 The biological activity of indiacens A (1) and B (2) was studied against various Gram-positive and Gram-negative bacteria as well as against fungi and yeasts (Table 2 and Table S3, Supporting Information). 1804
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20 min, rising to 65% B in 150 min; flow rate 30 mL/min; UV detection at 254 nm]. Compound 1 was collected at a retention time of 98 min and compound 2 at 110 min. The organic solvent of each fraction was evaporated, and the remaining aqueous layer extracted with ethyl acetate (three portions of 50 mL). The combined organic layers were washed with water (50 mL) and dried over Na2SO4 before drying in vacuo. The fraction containing 1 yielded 65 mg, and the fraction containing 2 yielded 55 mg. An 8 mg amount of the fraction with compound 1 was further purified by preparative TLC [20 × 20 cm, 0.25 mm, silica gel 60 F254 nm (Merck), DCM−MeOH, 98:2, UV detection 254 nm]. 1 had an Rf value of 0.23, and the UV active zone was scraped from the TLC plate. The silica gel was eluted with methanol, filtered through cotton, and dried in vacuo to yield 5.3 mg of 1. Analogously, 8 mg of the compound 2- containing fraction was purified by preparative TLC with an Rf value of 0.26 to yield 5.1 mg of 2. Indiacen A (1): white, amorphous solid (C14H13NO); [α]25D +2.4 (c 0.42, EtOH); UV (EtOH) λmax (log ε) 209 (4.298), 223 (4.374), 230 (4.365), 315 (4.368) nm; IR (KBr) ν 3166, 3109, 2999, 2954, 1641, 1608, 1599, 1405, 1294, 1145, 981, 772, 751, 645 cm−1; 1H and 13C NMR (acetone-d6, 400/100 MHz) see Table 1; HRESIMS m/z 212.1075 (calcd for C14H14NO, 212.1070). Indiacen B (2): white, amorphous solid (C14H12ClNO); [α]25D −0.57 (c 0.40, EtOH); UV (EtOH) λmax (log ε) 210 (4.316), 224 (4.364), 235 (4.372), 320 (4.428) nm; IR (KBr) ν 3191, 1636, 1622, 1511, 1460, 1405, 1372, 1298, 1149, 977, 795, 770, 645 cm−1; 1H and 13 C NMR (acetone-d6, 400/100 MHz) see Table 1; HRESIMS m/z 246.0685 (calcd for C14H13ClNO, 246.0680). Feeding Experiments. Sodium [1-13C]acetate [99% atom enriched (CIL); final concentration 0.05%], sodium [1,2-13C2]acetate [99% atom enriched, (CIL); final concentration 0.05%]; L [methyl-13C]methionine [98% atom enriched, (CIL); final concentration 0.02%], sodium [2-13C]propionate [99% atom enriched, (CIL); final concentration 0.04%], and [1,2-13C2]mevalonolactone [98% atom enriched (Sigma-Aldrich); final concentration 0.04%] were added in three portions (after 24, 48, and 72 h) each to 250 mL flasks with 100 mL of culture [K-medium: 0.2% potato-protein (EmslandGroup); 0.8% soluble starch; 0.4% probion (single cell protein, Hoechst); 0.2% yeast extract; 0.1% CaCl2·2H2O; 0.1%MgSO4·7H2O; 2.38% HEPES; 8 mg/mL Fe-EDTA; pH 7.5], supplemented with vitamins and 1% Amberlite XAD 16, respectively. Cultures were shaken at 160 rpm at 30 °C for one week. XAD was harvested with a sieve (200 μm), transferred to 50 mL Falcon tubes, and frozen (−20 °C). The XAD resin from each experiment was eluted with acetone (30 mL). The organic solvent was evaporated, and the aqueous residue was extracted with ethyl acetate (3 portions of 10 mL). The combined ethyl acetate layers of each feeding experiment were dried in vacuo. 1 was separated by RP-HPLC chromatography [column 250 × 21.2 mm Gemini C18 with SecurityGuard PREP (Phenomenex), 110 Å, 10 μm; solvent A: H2O−acetonitrile, 7:3; solvent B: H2O−acetonitrile, 6:4; gradient: 0% B to 75% B in 22.5 min, 75% B for 15 min; flow rate 20 mL/min; UV detection at 340 nm]. Compound 1 had a retention time of 25 min, while 2 eluted at 31 min. The organic solvent of each fraction was evaporated, and the water layer was extracted with ethyl acetate (2 portions of 10 mL). The indiacen A (1) and B (2) samples of each experiment were analyzed by NMR spectroscopy. The incorporation rate was calculated from integrals of the 1H signals of unlabeled and labeled exomethylene C-11, which were recognized from their large 1H,13C direct coupling constants (1JH,C = 156 Hz for 1 and 1JH,C = 196 Hz for 2). Indiacen A (1) showed 66% and indiacen B (2) 42% incorporation of labeled mevalonolactone, while feeding experiments with all other 13C-enriched precursors showed minimal incorporation. 1H and 13C spectra of labeled indiacens are shown in the Supporting Information. Biological Assays. A total of 2.0 and 10.0 μL of indiacens A (1) and B (2) (1 mg/mL) were tested against eight different Grampositive and five Gram-negative bacteria, four yeasts, and three fungi (see Table S3, Supporting Information). Minimal inhibition concentration (MIC) values were determined by serial dilution (150 μL) in 96-well plates for tissue cultures (TPP). Bacteria were
cultivated in EBS medium consisting of 0.5% peptone (Marcor M), 0.5% glucose, 0.1% meat extract, 0.1% yeast extract, and 50 mM HEPES at pH 7.0, and fungi/yeasts in MYC medium consisting of 1.0% phytone peptone, 1.0% glucose, and 1.19% HEPES at pH 7.0. The test organisms were cultivated at 30 °C and 160 rpm. In addition, a total of 3.0 μL of each of indiacens A (1) and B (2) (1 mg/mL) were tested against eukaryotic cells (mouse fibroblasts cell line; L929). MIC values were determined by serial dilution (60 μL) in 96-well plates for tissue cultures (Falcon). L929 cells were cultivated in DMEM medium (Lonza) + 10% FBS (Lonza) at 37 °C for five days in a CO2 incubator (5% CO2).
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ASSOCIATED CONTENT
S Supporting Information *
The complete NMR spectroscopic data of 1 and 2 measured in acetone-d6 are provided. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +49-681-30270201. Fax: +49-681-30270202. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank K. Harmrolfs for critical reading of the manuscript, D. Telkemeyer and S. Reinecke for technical support, W. Kessler and co-workers for large-scale fermentation, and C. Kakoschke for recording the NMR spectra.
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REFERENCES
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