- Email: [email protected]
Polyketides produced by Daldinia loculata cultured from Northern Manitoba
Tetrahedron Letters 52 (2011) 1697–1699
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Polyketides produced by Daldinia loculata cultured from Northern Manitoba Amy K. Nadeau, John L. Sorensen ⇑ Department of Chemistry, University of Manitoba, Winnipeg, Canada R3T 2N2
a r t i c l e
i n f o
Article history: Received 6 November 2010 Revised 28 January 2011 Accepted 31 January 2011 Available online 21 February 2011 Keywords: Polyketides Soil fungi Daldinia sp. Tyrosol
a b s t r a c t The fungus present in a soil sample collected from Northern Manitoba was cultured. The natural products produced by this organism were isolated and identified as the polyketides (1), (2), (3). A trace amount of the aromatic alcohol tyrosol (4) was also isolated. This organism was identified as Daldinia loculata by sequencing of the internally transcribed spacer region of the nuclear ribosomal DNA. The polyketides 1–3 were assayed against E. coli and it was observed that 1 displayed mild antibiotic activity. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Problems with resistance to current clinical antibiotics are driving the search for new molecules that can be used against infectious disease. There has been a recent resurgence in the exploration of novel natural products for development into clinical applications.1 One of the most promising applications of new natural products is the antibiotic secondary metabolites produced in fermentation culture by fungi. For example, the penicillin antibiotics, such as amoxicillin, are still produced commercially by the fermentation of Penicillium chrysogenum. A rich environment to explore for new strains of natural product producing fungi is in soil collected from remote environments. Soil fungi are a good source of new natural products as their environment is rich with competitors both within the soil and in the environment above. We have been examining the fungi present in soil that has been fortuitously sampled when new species have been added to the lichen herbarium at the University of Manitoba. Lichens are symbiotic associations between a mycobiont and a photosynthetic algal partner and are well known producers of biologically active natural products.2 The soil below these lichen represent an ecologically unique niche and have been collected from the remote Wapusk National Park in Northern Manitoba.3 Here we report the identification of the fungus Daldinia loculata cultured from a soil sample and the characterization of the compounds 1–4 from fermentation culture. This is the first report of the isolation of 1 and 4 from D. loculata fermentation cultures.
A monospore isolate of a filamentous fungus was cultured from soil collected with a lichen sample retrieved from Wapusk National Park, Northern Manitoba. These spores were used to inoculate Potato Dextrose Broth (PDB) fermentation cultures. After one week at 200 rpm and 30 °C the culture was harvested by filtration of the mycelia. The organism was identified as a strain of D. loculata by amplification and sequencing of the mitochondrial DNA. The fermentation broth was extracted with EtOAc and TLC and HPLC was used to detect the presence of secondary metabolites. Flash column chromatography on silica gel of the crude extract led to the purification of compounds 1–4 (Fig. 1). The structures for each compound were assigned by 1H and 13C NMR spectroscopy with connectivity established by two-dimensional techniques (COSY, HSQC, and HMBC). Assignments were confirmed by comparison with literature data when possible. The 1H NMR of 1, displayed a three proton spin system at d 6.42, 6.48, and 7.33 which suggested the presence of a tri-substituted aromatic system. A multiplet at 4.56 ppm (1H) suggested the presence of an oxygen atom in the structure while a two-proton spin system at 2.74 and 2.67 ppm indicated the presence of a diastereotopic methylene group. A doublet at 1.50 ppm (3H, J = 6.3 Hz) suggested that the methyl group was coupled to the multiplet at 4.65. These correlations were further confirmed by signals in the 2DCOSY spectra. An exchangeable proton d 11.70 (1H, s) indicated a phenolic proton hydrogen-bonded to a carbonyl group. A signal at 198.5 ppm in the 13C NMR also suggested the presence of a carbonyl group. The proton signals at 1.50 (2.74, 2.67) and 4.56 ppm correlated to carbon signals at 20.8, 43.8, and 73.8 ppm, respectively. In addition, correlations between protons signals at 6.42,
⇑ Corresponding author. Tel.: +1 204 474 9504; fax: +1 204 474 9504. E-mail address: [email protected] (J.L. Sorensen). 0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2011.01.150
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A. K. Nadeau, J. L. Sorensen / Tetrahedron Letters 52 (2011) 1697–1699
OH
O
OH
O
OH
OCH3 OH
O
O 1
2
HO 3
4
Figure 1. The structures of secondary metabolites isolated from Daldinia loculata.
6.48, and 7.35 ppm and carbon signals at 107.3, 108.1, and 138.1 ppm were observed. Additional signals in the 13C NMR at 108.1, 161.7, and 162.1 ppm were assigned to quaternary carbons. The NMR data for compound 1 was identical with the previously reported data for 5-hydroxy-2-methyl-chroman-4-one.4 The optical rotation was measured as ½a24 D = 1.19, suggesting that compound 1 is present as an excess of the (S) enantiomer. It has been previously reported that naturally produced 1 can exist as a pair of enantiomers and the absolute configuration of the purified enantiomers was determined by CD measurements and X-ray crystallography on the p-bromobenzoate derivative.5 The NMR data for compound 2 appeared quite similar to the data collected for compound 1 except that the presence of an additional carbon–carbon double bond was indicated. A singlet at 2.39 ppm (3H) suggested a methyl group attached to a sp2-hybrid carbon atom. This singlet was coupled to a carbon signal at 20.8 ppm. A singlet at 6.12 ppm (1H) coupled to a carbon signal at 109.1 ppm also suggested the presence of a carbon–carbon double bond in 2. A three-proton spin system 6.77, 6.86, and 7.49 ppm suggested a tri-substituted aromatic ring. These signals were coupled to carbons at 111.2, 106.8, and 135.3 ppm further supporting this conclusion. An exchangeable proton at 12.54 ppm (1H) suggested the presence of a phenolic substituent adjacent to a carbonyl group (183.1 ppm). Signals at 110.1, 156.8, 160.8, and 167.7 ppm were assigned to quaternary carbons. The NMR data for 2 is consistent with previously reported data for peperovulcanone A which contains a similar structural fragment.6 The NMR data for compound 3, a naphthalene derivative, was compared to literature values for 8-methoxynaphthalen-1-ol.7,8 A singlet at 4.05 ppm (3H) was assigned to the methoxy group and the singlet at 9.37 ppm to the exchangeable proton of the phenol. Two protons appeared at 6.78 and 6.93 ppm while four signals appeared in the range from 7.29 to 7.44 ppm. In the 13C NMR, the signal at 56.2 ppm was assigned to the methyl carbon while 10 other signals above 100 ppm indicated the naphthalene ring. The HSQC spectra displayed a correlation between the proton at 4.05 ppm and the carbon at 56.2 ppm, while protons at 6.78 and 6.93 ppm displayed correlations to carbon signals at 104.0 and 110.6 ppm, respectively. The protons at 7.29, 7.32, 7.36, and 7.44 ppm correlated to carbons at 125.8, 118.0, 125.8, and 121.9 ppm, respectively. The 13C signals at 156.3, 154.6, 136.9, and 115.2 ppm were assigned to quaternary carbons. Tyrosol (4) displayed signals in the 1H NMR at 7.10 (2H) and 6.78 (2H) ppm characteristic of an aromatic ring disubstituted in a para position. At 3.49 ppm (1H) a singlet indicated a hydroxyl proton. The triplet signal at 3.83 ppm (2H, J = 6.5 Hz) corresponded to the methylene bearing the hydroxyl group. Another triplet at 2.80 ppm (2H, J = 6.5 Hz) corresponded to the methylene linked to the aromatic ring. The NMR data collected on the tyrosol (4) purified from the fermentation culture was in complete agreement with the data collected on a standard commercial sample of tyrosol.9 The biological activity of 1–3 has been previously reported in an assay against the fungi Microbotryum violaceum and Septoria tritici, where compound 1 was shown to have twice as much activity as compound 3.5 However, in their assay against the bacterium,
Bacillus meaterium, none of compounds 1–3 showed activity. We assayed compounds 1–3 for antibiotic activity against DH5a E. coli cells at a concentration of 1 mg/mL (ca. 5 mM) using an agar overlay assay. At this concentration a mild antibiotic activity was observed for compound 1 and less so for 3 while 2 appeared inactive. The results reported here are consistent with the previous reported fungal assay.5 The crude EtOAc fermentation extract was also assayed at a concentration of 10 mg/mL and displayed mild antibiotic activity. The biological activity of 4 is well established and was not assayed here. A summary of the biological activity assays is given in Table S1 in the Supporting Information. Compounds 1–3 appear to be of polyketide origin, although it appears likely that 1 is derived from 2 by an enzymatic reduction. Compound 1 has not been previously reported from D. loculata but has been reported from the related species D. concentrica10–12, as well as the plant pathogen Phialophora gregata4, and a Nodulisporum species.5,13 Compounds 1 and 2 have been reported also from Nodulisporum species.5 Other ascomycete Xylariaceae family members have also been shown to produce 1 and 2.14 Compound 3, a naphthalene derivative, may be produced as a side product of melanin biosynthesis as the related compound 1,8-dihydroxynaphthalene is the general melanin precursor in ascomycetes.15 Tyrosol (4) is a well known antioxidant plant metabolite and is an important constituent of olive oil.16 Tyrosol (4) has been recently reported as a product of fungal fermentation.9
3. Experimental Solvents and media components were purchased from Sigma– Aldrich Inc., St Louis, MO, USA. All solvents were HPLC grade and used as such. Nuclear Magnetic Resonance (NMR) experiments were obtained on a Bruker AMX500 or a Bruker AMX300 spectrometer. Flash chromatography was performed using silica gel (230–430 mesh, 60 Å, Fisher Scientific, Fair Lawn, NJ, USA) and elution of solvent with positive air pressure. A UnicoÒ 1100 spectrophotometer was used to read E. coli cell density at 600 nm. For HPLC analysis, a Waters HPLC Separations Module 2695, combined with a PDA Detector Model 2996 was used. The column was a lBondapakÒ Waters C18 (3.9 300 mm) column particle diameter of 15–20 lm, with 125 Å pores. The flow rate was 1 mL/min. The eluent was monitored continuously at 210–600 nm and HPLC traces were displayed at 220 nm and 254 nm. The gradient was held at 20% acetonitrile in 0.075% aqueous trifluoracetic acid for 10 min then a linear gradient to 80% acetonitrile and held at that composition for 20 min. followed by a linear gradient back to 20% acetonitrile for 10 min and held there for 10 min. The total run time was 60 min. A soil sample (ca. 0.5 g) was isolated from the base of the thallus of the lichen collected June 23, 2005 from Wapusk National Park (University of Manitoba accession number 006641 MPN). A portion of the soil sample was surface sterilized with several alternate washes of 3% sodium hypochlorite and sterile H2O, with a final rinse of copious amounts of sterile H2O. The sterilized soil sample was placed on several potato dextrose agar (PDA) plates and incubated for one week at 25 °C. One of the plates displayed a slow
A. K. Nadeau, J. L. Sorensen / Tetrahedron Letters 52 (2011) 1697–1699
growing dark fungus with a concentric ring pattern. A subculture of this fungus was grown on PDA resulting in a single spore culture. Potato dextrose broth (PDB) fermentation cultures (2 1 L each in an unbaffled 2 L Erlenmeyer flask) were inoculated with spores from this sub-culture and placed in a shaker at 30 °C and 200 rpm for one week under ambient light. The fermentation broth was filtered to remove mycelia, combined and extracted with EtOAc (2 500 mL). The organic layer was dried (Na2SO4), filtered and evaporated to provide 128 mg of crude extract. The crude extract was then fractionated by flash column chromatography over silica gel using a gradient of hexane– EtOAc (9:1) to 100% EtOAC. Fractions were analyzed by TLC using 1% MeOH in CH2Cl2 as the eluent and fractions displaying similar Rf values were pooled together and evaporated. Further analysis by TLC indicated the presence of four pure compounds with Rf values of 0.81, 0.76, 0.64, and 0.31. Compounds 1 and 2 were analyzed by HPLC, and showed retention times of 16.5 and 15.7 min, respectively. The identities of these compounds were confirmed by both 1 H and 13C NMR characterization as reported in Section 2. The complete characterization data, including 1H and 13C and 2D NMR spectra are included in the Supporting Information. Biological assays of the purified compounds were carried out as follows. LB media (5 mL) was inoculated with 200 lL of frozen stock E. coli, and incubated at 37 °C and 200 rpm overnight. The cells were diluted with sterile LB broth until they gave an optical density reading of 1.00 at 600 nm. LB agar plates (60 15 mm) were prepared and allowed to solidify. LB half strength agar was inoculated with E. coli (OD = 1.00) 200 lL per 1 mL of LB half strength agar and mixed thoroughly. On top of the solid LB agar 3 mL of the inoculated half strength agar was poured and allowed to solidify. Compounds (1–3) were dissolved in acetone at a concentration of 1 mg/mL and 10 lL of each solution was placed on separate plates. The crude extracts from the fermentation cultures were assayed at a concentration of 10 mg/mL. Control plates included an acetone solvent control, streptomycin positive control and an undisturbed E. coli negative control plate. All plates were prepared in duplicate and then incubated at 37 °C overnight. Biological activity was interpreted as a zone of clearing where the compound was applied. The internal transcribed spacer (ITS) in the mtDNA was sequenced as described below. The DNA was extracted from the mycelia using a modification of a previously reported method.17 The primer pair 1780-50 (SSU rDNA)18 and ITS2-kL-30 (ITS rDNA)18 were used for DNA amplification, and the DNA extracted from the agarose gel. Sequencing generated a nucleotide sequence of 1039 base pairs. The sequence was aligned using a BLAST search19 and was identified as a strain of D. loculata based upon homology (>99%) to several strains. The amplified nucleotide sequence is given in full in the Supporting Information and has been submitted to GenBank where it was assigned the accession number HQ671314.
1699
4. Conclusions We report here for the first time the isolation of 1 and 4 from fermentation cultures of a strain of D. loculata present in soil collected from a remote region of Northern Manitoba. In addition we report the isolation of the polyketides 2 and 3 from the same fermentation cultures. Compound 1 displayed mild antibiotic activity and is perhaps the reason that this strain of D. loculata produces 1 in fermentation culture. We have demonstrated that strains of known fungi, when isolated from unusual habitats, can display an altered profile of secondary metabolites. This indicates that further searches of organisms from remote locations such as Wapusk National Park may yield promising lead compounds for development into clinical antibiotics. Acknowledgments Access to the University of Manitoba Lichen Herbarium was provided by Dr. Michele Piercey-Normore and is gratefully acknowledged. This work was partially funded by the Natural Sciences and Engineering Research Council (NSERC) and the University of Manitoba. Dr. Harry Duckworth, University of Manitoba, is also gratefully acknowledged for his support of this work. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tetlet.2011.01.150. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Li, J. W.-H.; Vederas, J. C. Science 2009, 325, 161–165. Stocker-Worgotter, E. Nat. Prod. Rep. 2008, 25, 188–200. Piercey-Normore, M. D. Botany 2010, 88, 923–929. Gray, L. M.; Gardner, H. W.; Weisleder, D.; Leib, M. Phytochemistry 1999, 50, 1337–1340. Dai, J.; Krohn, K.; Flörke, U.; Draeger, S.; Schulz, B.; Kiss-Szikaszai, A.; Antus, S.; Kurtán, T.; van Ree, T. Eur. J. Org. Chem. 2006, 15, 3498–3506. Cheng, M.; Chen, I. J. Chil. Chem. Soc. 2008, 53, 1539–1541. Anke, H.; Stadler, V.; Mayer, A.; Stemer, O. Can. J. Botany 1995, 73, 932–939 (Suppl. 1, Sect. E–H, Fifth International Mycological Congress, Sect. E–H, 1994). Allport, D. C.; Bu’Lock, J. D. J. Chem. Soc. 1960, 654. Tyagunova, Y.; Sorensen, J. L. Nat. Prod. Commun. 2010, 5, 81–84. Want, F.; Liu, J. Helv. Chim. Acta 2004, 87, 2131–2134. Wu, Z.; Li, D.; Chen, Y.; Zhang, W. Helv. Chim. Acta 2010, 93, 920–924. Stadler, M.; Wollweber, H. Mycotaxon 2001, 76, 379–429. Anderson, J. R.; Edwards, R. L. J. Chem. Soc., Perkin Trans. 1 1983, 2185–2192. Stadler, M.; Baumgartner, M.; Grothe, T.; Andrea, M.; Seip, S.; Hartmund, W. Phytochemistry 2001, 56, 787–793. Kiritsakis, A. K. J. Am. Oil Chem. Soc. 1998, 75, 673–681. Grube, M.; DePriest, P. T.; Gargas, A.; Hafellner, J. Mycol. Res. 1995, 99, 1321– 1324. Gargas, A.; Taylor, J. W. Mycologia 1992, 84, 589–592. White, T. J.; Bruns, T.; Lee, S.; Taylor, J. PCR Protocols: A Guide to Methods and Applications 1990, 315–322. Zhang, Z.; Schwartz, S.; Wagner, L.; Miller, W. J. Comput. Biol. 2000, 7, 203–214.
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Polyketides produced by Daldinia loculata cultured from Northern Manitoba Amy K. Nadeau, John L. Sorensen ⇑ Department of Chemistry, University of Manitoba, Winnipeg, Canada R3T 2N2
a r t i c l e
i n f o
Article history: Received 6 November 2010 Revised 28 January 2011 Accepted 31 January 2011 Available online 21 February 2011 Keywords: Polyketides Soil fungi Daldinia sp. Tyrosol
a b s t r a c t The fungus present in a soil sample collected from Northern Manitoba was cultured. The natural products produced by this organism were isolated and identified as the polyketides (1), (2), (3). A trace amount of the aromatic alcohol tyrosol (4) was also isolated. This organism was identified as Daldinia loculata by sequencing of the internally transcribed spacer region of the nuclear ribosomal DNA. The polyketides 1–3 were assayed against E. coli and it was observed that 1 displayed mild antibiotic activity. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
Problems with resistance to current clinical antibiotics are driving the search for new molecules that can be used against infectious disease. There has been a recent resurgence in the exploration of novel natural products for development into clinical applications.1 One of the most promising applications of new natural products is the antibiotic secondary metabolites produced in fermentation culture by fungi. For example, the penicillin antibiotics, such as amoxicillin, are still produced commercially by the fermentation of Penicillium chrysogenum. A rich environment to explore for new strains of natural product producing fungi is in soil collected from remote environments. Soil fungi are a good source of new natural products as their environment is rich with competitors both within the soil and in the environment above. We have been examining the fungi present in soil that has been fortuitously sampled when new species have been added to the lichen herbarium at the University of Manitoba. Lichens are symbiotic associations between a mycobiont and a photosynthetic algal partner and are well known producers of biologically active natural products.2 The soil below these lichen represent an ecologically unique niche and have been collected from the remote Wapusk National Park in Northern Manitoba.3 Here we report the identification of the fungus Daldinia loculata cultured from a soil sample and the characterization of the compounds 1–4 from fermentation culture. This is the first report of the isolation of 1 and 4 from D. loculata fermentation cultures.
A monospore isolate of a filamentous fungus was cultured from soil collected with a lichen sample retrieved from Wapusk National Park, Northern Manitoba. These spores were used to inoculate Potato Dextrose Broth (PDB) fermentation cultures. After one week at 200 rpm and 30 °C the culture was harvested by filtration of the mycelia. The organism was identified as a strain of D. loculata by amplification and sequencing of the mitochondrial DNA. The fermentation broth was extracted with EtOAc and TLC and HPLC was used to detect the presence of secondary metabolites. Flash column chromatography on silica gel of the crude extract led to the purification of compounds 1–4 (Fig. 1). The structures for each compound were assigned by 1H and 13C NMR spectroscopy with connectivity established by two-dimensional techniques (COSY, HSQC, and HMBC). Assignments were confirmed by comparison with literature data when possible. The 1H NMR of 1, displayed a three proton spin system at d 6.42, 6.48, and 7.33 which suggested the presence of a tri-substituted aromatic system. A multiplet at 4.56 ppm (1H) suggested the presence of an oxygen atom in the structure while a two-proton spin system at 2.74 and 2.67 ppm indicated the presence of a diastereotopic methylene group. A doublet at 1.50 ppm (3H, J = 6.3 Hz) suggested that the methyl group was coupled to the multiplet at 4.65. These correlations were further confirmed by signals in the 2DCOSY spectra. An exchangeable proton d 11.70 (1H, s) indicated a phenolic proton hydrogen-bonded to a carbonyl group. A signal at 198.5 ppm in the 13C NMR also suggested the presence of a carbonyl group. The proton signals at 1.50 (2.74, 2.67) and 4.56 ppm correlated to carbon signals at 20.8, 43.8, and 73.8 ppm, respectively. In addition, correlations between protons signals at 6.42,
⇑ Corresponding author. Tel.: +1 204 474 9504; fax: +1 204 474 9504. E-mail address: [email protected] (J.L. Sorensen). 0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2011.01.150
1698
A. K. Nadeau, J. L. Sorensen / Tetrahedron Letters 52 (2011) 1697–1699
OH
O
OH
O
OH
OCH3 OH
O
O 1
2
HO 3
4
Figure 1. The structures of secondary metabolites isolated from Daldinia loculata.
6.48, and 7.35 ppm and carbon signals at 107.3, 108.1, and 138.1 ppm were observed. Additional signals in the 13C NMR at 108.1, 161.7, and 162.1 ppm were assigned to quaternary carbons. The NMR data for compound 1 was identical with the previously reported data for 5-hydroxy-2-methyl-chroman-4-one.4 The optical rotation was measured as ½a24 D = 1.19, suggesting that compound 1 is present as an excess of the (S) enantiomer. It has been previously reported that naturally produced 1 can exist as a pair of enantiomers and the absolute configuration of the purified enantiomers was determined by CD measurements and X-ray crystallography on the p-bromobenzoate derivative.5 The NMR data for compound 2 appeared quite similar to the data collected for compound 1 except that the presence of an additional carbon–carbon double bond was indicated. A singlet at 2.39 ppm (3H) suggested a methyl group attached to a sp2-hybrid carbon atom. This singlet was coupled to a carbon signal at 20.8 ppm. A singlet at 6.12 ppm (1H) coupled to a carbon signal at 109.1 ppm also suggested the presence of a carbon–carbon double bond in 2. A three-proton spin system 6.77, 6.86, and 7.49 ppm suggested a tri-substituted aromatic ring. These signals were coupled to carbons at 111.2, 106.8, and 135.3 ppm further supporting this conclusion. An exchangeable proton at 12.54 ppm (1H) suggested the presence of a phenolic substituent adjacent to a carbonyl group (183.1 ppm). Signals at 110.1, 156.8, 160.8, and 167.7 ppm were assigned to quaternary carbons. The NMR data for 2 is consistent with previously reported data for peperovulcanone A which contains a similar structural fragment.6 The NMR data for compound 3, a naphthalene derivative, was compared to literature values for 8-methoxynaphthalen-1-ol.7,8 A singlet at 4.05 ppm (3H) was assigned to the methoxy group and the singlet at 9.37 ppm to the exchangeable proton of the phenol. Two protons appeared at 6.78 and 6.93 ppm while four signals appeared in the range from 7.29 to 7.44 ppm. In the 13C NMR, the signal at 56.2 ppm was assigned to the methyl carbon while 10 other signals above 100 ppm indicated the naphthalene ring. The HSQC spectra displayed a correlation between the proton at 4.05 ppm and the carbon at 56.2 ppm, while protons at 6.78 and 6.93 ppm displayed correlations to carbon signals at 104.0 and 110.6 ppm, respectively. The protons at 7.29, 7.32, 7.36, and 7.44 ppm correlated to carbons at 125.8, 118.0, 125.8, and 121.9 ppm, respectively. The 13C signals at 156.3, 154.6, 136.9, and 115.2 ppm were assigned to quaternary carbons. Tyrosol (4) displayed signals in the 1H NMR at 7.10 (2H) and 6.78 (2H) ppm characteristic of an aromatic ring disubstituted in a para position. At 3.49 ppm (1H) a singlet indicated a hydroxyl proton. The triplet signal at 3.83 ppm (2H, J = 6.5 Hz) corresponded to the methylene bearing the hydroxyl group. Another triplet at 2.80 ppm (2H, J = 6.5 Hz) corresponded to the methylene linked to the aromatic ring. The NMR data collected on the tyrosol (4) purified from the fermentation culture was in complete agreement with the data collected on a standard commercial sample of tyrosol.9 The biological activity of 1–3 has been previously reported in an assay against the fungi Microbotryum violaceum and Septoria tritici, where compound 1 was shown to have twice as much activity as compound 3.5 However, in their assay against the bacterium,
Bacillus meaterium, none of compounds 1–3 showed activity. We assayed compounds 1–3 for antibiotic activity against DH5a E. coli cells at a concentration of 1 mg/mL (ca. 5 mM) using an agar overlay assay. At this concentration a mild antibiotic activity was observed for compound 1 and less so for 3 while 2 appeared inactive. The results reported here are consistent with the previous reported fungal assay.5 The crude EtOAc fermentation extract was also assayed at a concentration of 10 mg/mL and displayed mild antibiotic activity. The biological activity of 4 is well established and was not assayed here. A summary of the biological activity assays is given in Table S1 in the Supporting Information. Compounds 1–3 appear to be of polyketide origin, although it appears likely that 1 is derived from 2 by an enzymatic reduction. Compound 1 has not been previously reported from D. loculata but has been reported from the related species D. concentrica10–12, as well as the plant pathogen Phialophora gregata4, and a Nodulisporum species.5,13 Compounds 1 and 2 have been reported also from Nodulisporum species.5 Other ascomycete Xylariaceae family members have also been shown to produce 1 and 2.14 Compound 3, a naphthalene derivative, may be produced as a side product of melanin biosynthesis as the related compound 1,8-dihydroxynaphthalene is the general melanin precursor in ascomycetes.15 Tyrosol (4) is a well known antioxidant plant metabolite and is an important constituent of olive oil.16 Tyrosol (4) has been recently reported as a product of fungal fermentation.9
3. Experimental Solvents and media components were purchased from Sigma– Aldrich Inc., St Louis, MO, USA. All solvents were HPLC grade and used as such. Nuclear Magnetic Resonance (NMR) experiments were obtained on a Bruker AMX500 or a Bruker AMX300 spectrometer. Flash chromatography was performed using silica gel (230–430 mesh, 60 Å, Fisher Scientific, Fair Lawn, NJ, USA) and elution of solvent with positive air pressure. A UnicoÒ 1100 spectrophotometer was used to read E. coli cell density at 600 nm. For HPLC analysis, a Waters HPLC Separations Module 2695, combined with a PDA Detector Model 2996 was used. The column was a lBondapakÒ Waters C18 (3.9 300 mm) column particle diameter of 15–20 lm, with 125 Å pores. The flow rate was 1 mL/min. The eluent was monitored continuously at 210–600 nm and HPLC traces were displayed at 220 nm and 254 nm. The gradient was held at 20% acetonitrile in 0.075% aqueous trifluoracetic acid for 10 min then a linear gradient to 80% acetonitrile and held at that composition for 20 min. followed by a linear gradient back to 20% acetonitrile for 10 min and held there for 10 min. The total run time was 60 min. A soil sample (ca. 0.5 g) was isolated from the base of the thallus of the lichen collected June 23, 2005 from Wapusk National Park (University of Manitoba accession number 006641 MPN). A portion of the soil sample was surface sterilized with several alternate washes of 3% sodium hypochlorite and sterile H2O, with a final rinse of copious amounts of sterile H2O. The sterilized soil sample was placed on several potato dextrose agar (PDA) plates and incubated for one week at 25 °C. One of the plates displayed a slow
A. K. Nadeau, J. L. Sorensen / Tetrahedron Letters 52 (2011) 1697–1699
growing dark fungus with a concentric ring pattern. A subculture of this fungus was grown on PDA resulting in a single spore culture. Potato dextrose broth (PDB) fermentation cultures (2 1 L each in an unbaffled 2 L Erlenmeyer flask) were inoculated with spores from this sub-culture and placed in a shaker at 30 °C and 200 rpm for one week under ambient light. The fermentation broth was filtered to remove mycelia, combined and extracted with EtOAc (2 500 mL). The organic layer was dried (Na2SO4), filtered and evaporated to provide 128 mg of crude extract. The crude extract was then fractionated by flash column chromatography over silica gel using a gradient of hexane– EtOAc (9:1) to 100% EtOAC. Fractions were analyzed by TLC using 1% MeOH in CH2Cl2 as the eluent and fractions displaying similar Rf values were pooled together and evaporated. Further analysis by TLC indicated the presence of four pure compounds with Rf values of 0.81, 0.76, 0.64, and 0.31. Compounds 1 and 2 were analyzed by HPLC, and showed retention times of 16.5 and 15.7 min, respectively. The identities of these compounds were confirmed by both 1 H and 13C NMR characterization as reported in Section 2. The complete characterization data, including 1H and 13C and 2D NMR spectra are included in the Supporting Information. Biological assays of the purified compounds were carried out as follows. LB media (5 mL) was inoculated with 200 lL of frozen stock E. coli, and incubated at 37 °C and 200 rpm overnight. The cells were diluted with sterile LB broth until they gave an optical density reading of 1.00 at 600 nm. LB agar plates (60 15 mm) were prepared and allowed to solidify. LB half strength agar was inoculated with E. coli (OD = 1.00) 200 lL per 1 mL of LB half strength agar and mixed thoroughly. On top of the solid LB agar 3 mL of the inoculated half strength agar was poured and allowed to solidify. Compounds (1–3) were dissolved in acetone at a concentration of 1 mg/mL and 10 lL of each solution was placed on separate plates. The crude extracts from the fermentation cultures were assayed at a concentration of 10 mg/mL. Control plates included an acetone solvent control, streptomycin positive control and an undisturbed E. coli negative control plate. All plates were prepared in duplicate and then incubated at 37 °C overnight. Biological activity was interpreted as a zone of clearing where the compound was applied. The internal transcribed spacer (ITS) in the mtDNA was sequenced as described below. The DNA was extracted from the mycelia using a modification of a previously reported method.17 The primer pair 1780-50 (SSU rDNA)18 and ITS2-kL-30 (ITS rDNA)18 were used for DNA amplification, and the DNA extracted from the agarose gel. Sequencing generated a nucleotide sequence of 1039 base pairs. The sequence was aligned using a BLAST search19 and was identified as a strain of D. loculata based upon homology (>99%) to several strains. The amplified nucleotide sequence is given in full in the Supporting Information and has been submitted to GenBank where it was assigned the accession number HQ671314.
1699
4. Conclusions We report here for the first time the isolation of 1 and 4 from fermentation cultures of a strain of D. loculata present in soil collected from a remote region of Northern Manitoba. In addition we report the isolation of the polyketides 2 and 3 from the same fermentation cultures. Compound 1 displayed mild antibiotic activity and is perhaps the reason that this strain of D. loculata produces 1 in fermentation culture. We have demonstrated that strains of known fungi, when isolated from unusual habitats, can display an altered profile of secondary metabolites. This indicates that further searches of organisms from remote locations such as Wapusk National Park may yield promising lead compounds for development into clinical antibiotics. Acknowledgments Access to the University of Manitoba Lichen Herbarium was provided by Dr. Michele Piercey-Normore and is gratefully acknowledged. This work was partially funded by the Natural Sciences and Engineering Research Council (NSERC) and the University of Manitoba. Dr. Harry Duckworth, University of Manitoba, is also gratefully acknowledged for his support of this work. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.tetlet.2011.01.150. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
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