Article pubs.acs.org/jnp
Ramariolides A−D, Antimycobacterial Butenolides Isolated from the Mushroom Ramaria cystidiophora Ryan M. Centko,† Santiago Ramón-García,‡,§ Terry Taylor,‡ Brian O. Patrick,† Charles J. Thompson,‡,§ Vivian P. Miao,‡ and Raymond J. Andersen*,† †
Departments of Chemistry and Earth, Ocean & Atmospheric Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada ‡ Department of Microbiology and Immunology and §Centre for Tuberculosis Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada S Supporting Information *
ABSTRACT: Four butenolides, ramariolides A−D (1−4), have been isolated from the fruiting bodies of the coral mushroom Ramaria cystidiophora. Their structures were elucidated by analysis of 1D and 2D NMR data and a single-crystal X-ray diffraction analysis of 1, and their absolute configurations were established using Mosher’s method. The major metabolite, ramariolide A (1), which contains an unusual spiro oxiranebutenolide moiety, showed in vitro antimicrobial activity against Mycobacterium smegmatis and Mycobacterium tuberculosis.
T
uberculosis (TB), which is caused by Mycobacterium tuberculosis, is one of the most devastating diseases in the world. It has been estimated that in 2010 TB was responsible for 1.2−1.5 million deaths worldwide.1 The recommended regimen for treatment is lengthy and severely compromised by the emergence of drug-resistant strains of the pathogen as well as co-infection with HIV, and, therefore, new treatment options are urgently needed. Fungi are a proven source of therapeutic small molecules such as penicillin and cyclosporin, and they continue to be a source of novel natural products.2,3 However, many species of uncultivated mushroom-producing fungi may be overlooked because they exist for the greater part of their life cycle as inconspicuous microscopic cells in the soil, where they are components of complex mycorrhizal (nutritionally interlinked) networks involving higher plants. The macroscopic fruiting bodies that are produced by these fungi are typically seasonal and subject to weather. Collecting significant biomass of rare mushrooms is often challenging. Nonetheless, the mushroom stage represents an opportunity to access some of these fungi, and chemical characterization of the mushrooms reveals the metabolic potential of a variety of developmental and physiological tissue types, offering an opportunity to discover new biologically active natural products. Members of the genus Ramaria are known as “coral fungi”. They number about 500 species worldwide, and many are considered mycorrhizal.4 In the Pacific Northwest region of North America, species richness has been strongly associated with old growth forests.5 Recent research has shed light on the molecular phylogenetics of these fungi,6 but little is known about their chemistry. Only a ceramide, several sterols,7 and the siderophore pistillarin8 have been described from the Ramaria to date. Extracts of a selection of Ramaria species from © 2012 American Chemical Society and American Society of Pharmacognosy
southern British Columbia have been examined as part of a screening program of uncultivated mushrooms for antimycobacterial activities against the model organism Mycobacterium smegmatis. Herein, we describe the isolation and characterization of a new group of butenolides, ramariolides A to D (1− 4), from Ramaria cystidiophora. Ramariolide A (1), which contains a spiro oxiranebutenolide moiety, showed in vitro antimicrobial activity against M. smegmatis and M. tuberculosis.
■
RESULTS AND DISCUSSION An EtOAc extract of the coral mushroom R. cystidiophora showed promising antibacterial activity against M. smegmatis. Fractionation of the R. cystidiophora extract using Sephadex LH20 chromatography (eluent: 1:1 CH2Cl2/MeOH), followed by sequential C18 and C8 reversed-phase HPLC purifications of the active fractions, led to the isolation of ramariolides A (1) to D (4). Ramariolide A (1) was isolated as an optically active, white crystalline solid that gave a [M − H]− ion in the HRESIMS at m/z 281.1769, appropriate for a molecular formula of C16H26O4 requiring four sites of unsaturation. The 13C NMR spectrum contained 16 resolved carbon resonances (2 × C, 4 × CH, 9 × CH2, and 1 × CH3), in agreement with the ESIHRMS measurement, and the DEPT and HSQC spectra identified 25 hydrogens attached to carbon, indicating that there was one exchangeable hydrogen atom. Resonances in the 13C NMR spectrum could be assigned to a disubstituted alkene [δ 126.7 (C-2) and δ 149.6 (C-3)] and an unsaturated ester or carboxylic acid carbonyl [δ 168.9 (C-1)]. The absence of 13C Received: September 11, 2012 Published: December 3, 2012 2178
dx.doi.org/10.1021/np3006277 | J. Nat. Prod. 2012, 75, 2178−2182
Journal of Natural Products
Article
this assignment, the H-7/H-7′ methylene resonances (δ 1.47 and 1.63) showed HMBC correlations to C-8 (δ 25.6) and to one member (C-9) of a complex of resonances between δ 29.9 and 30.1, all having chemical shifts typical of the methylene carbons of a saturated linear alkyl chain. Ramariolide A (1) gave crystals from CH2Cl2 that were subjected to single-crystal X-ray diffraction analysis. An ORTEP diagram generated from the analysis is shown in Figure 2. Due
NMR evidence for additional unsaturated functionalities indicated that 1 was bicyclic. A pair of olefinic methine doublets at δ 6.34 (H-2, J = 5.6 Hz) and δ 7.55 (H-3, J = 5.6 Hz) in the 1H NMR spectrum showed COSY correlations to each other and HMBC correlations to the carbonyl resonance at δ 168.9 (C-1) and a ketal resonance at δ 90.3 (C-4), consistent with fragment A shown in Figure 1. 1H NMR resonances at δ 3.75 (H-5) and δ
Figure 2. ORTEP diagram for ramariolide A (1).
to the weakly scattering crystal, an unrestrained anisotropic refinement of the structure was not possible; however the atomic connectivity, particularly with respect to the rigid spiro oxiranebutenolide fragment of the molecule, is not in doubt. This confirmed the constitution determined by the NMR analysis outlined above and also provided the 4R*,5R*,6R* relative configurations shown in the ORTEP diagram. A Mosher ester analysis of 1 (Supporting Information) revealed that C-6 had the S absolute configuration, and, therefore, ramariolide A has the 4S,5S,6S absolute configuration.9 Ramariolide B (2) was isolated as an optically active oil that gave a [M + Na]+ ion in the HRESIMS at m/z 305.1721, appropriate for a molecular formula of C16H26O4, indicating it was an isomer of 1. The 13C NMR spectrum of 2 contained 16 resolved resonances (2 × C, 4 × CH, 9 × CH2, 1 × CH3), in agreement with the HRESIMS measurement, and the HSQC spectrum identified 25 hydrogen atoms attached to carbon, requiring one exchangeable hydrogen atom as in 1. The 1H and 13 C NMR spectra obtained for 2 showed a strong resemblance to the spectra obtained for 1. COSY and HMBC correlations observed for 2 confirmed the presence of the same butenolide and linear C-8 to C-16 alkyl chain fragments present in 1. The COSY data also identified a partial linear spin system consisting of vicinal oxymethine protons [δ 3.81 (H-5), δ 4.32 (H-6)] and an aliphatic methylene [δ 1.38 (H-7), δ 1.47 (H-7′)] similar to the H-5 to H-7/H-7′ spin system in 1 (Figure 3). However, the ketal resonance at δ 112.0 (C-4) and oxymethine carbon resonances at δ 74.4 (C-5) and δ 90.8 (C-6), and their attached proton resonances at δ 3.81 (H-5) and δ 4.32 (H-6), were shifted downfield compared with the corresponding resonances
Figure 1. Fragments A, B, and C constructed using gHMBC and gCOSY data.
4.05 (H-6), which showed HSQC correlations to carbon resonances at δ 64.3 (C-5) and δ 67.9 (C-6), respectively, were assigned to a pair of oxymethine carbons. The H-6 resonance at δ 4.05 showed COSY correlations to the H-5 resonance at δ 3.75, to an exchangeable resonance at δ 1.99 (OH-6), and to a pair of geminal methylene resonances at δ 1.63 (H-7) and δ 1.47 (H-7′), as indicated in fragment B in Figure 1. HMBC correlations observed between H-5 (δ 3.75) and C-6 (δ 67.9), between OH-6 (δ 1.99) and both C-5 (δ 64.3) and C-6 (δ 67.9), and between H-6 (δ 4.05) and C-7 (δ 34.0) and C-8 (δ 32.0) supported the structure assigned to fragment B. HMBC correlations observed between both H-5 (δ 3.75) and H-6 (δ 4.05) and the ketal carbon C-4 (δ 90.3) showed that fragment A was linked to fragment B via a C-4/C-5 bond to give expanded fragment C. A C-1 to C-4 butenolide and an oxirane linkage between the oxymethine C-5 and the C-4 ketal provided the two rings required by the molecular formula. The remaining fragment of 1 had to be saturated and account for C8H17. Only one methyl resonance, a triplet at δ 0.88 (Me-16), was observed in the 1H NMR spectrum of 1, indicating that C8 had to be one terminus of a nine-carbon linear alkyl chain attached to C-7 to complete the structure. In agreement with
Figure 3. COSY and HMBC correlations for ramariolide B (2). 2179
dx.doi.org/10.1021/np3006277 | J. Nat. Prod. 2012, 75, 2178−2182
Journal of Natural Products
Article
the 10 -carbon side chain in 4. Again, we have assumed the C-6 has the S configuration in ramariolide D (4) as in 1. The minimal inhibitory concentration (MIC) of ramariolide A (1) determined by broth dilution against M. smegmatis was 8 μg/mL, and the minimum bactericidal concentration (MBC) to MIC ratio was 1, indicating that the effect was bactericidal (Figure 4A). For comparison, isoniazid, currently a first-line
in the NMR spectra of 1. In addition, the exchangeable proton resonance at δ 2.11 (OH-5) showed COSY correlations to H-5, suggesting a hydroxy substituent at C-5 in 2 instead of at C-6 as in 1. The presence of the C-5 hydroxy substituent was confirmed by the observation of HMBC correlations between OH-5 (δ 2.11) and C-4 (δ 112.0), C-5 (δ 74.4), and C-6 (δ 90.8). An ether linkage between C-6 and C-4 (Figure 3), which formed an oxetane in 2, in place of the oxirane in 1, provided the second ring required by the molecular formula. The change in ring size accounts for the downfield trend in chemical shifts of the 1H and 13C resonances assigned to the C-4 to C-6 region of 2 compared with the corresponding assignments for 1. A strong 1D NOESY correlation observed between H-3 (δ 5.37) and H-5 (δ 3.81) showed that C-3 and H-5 were on the same face of the oxetane ring in 2. A weak 1D NOESY signal was observed between H-5 (δ 3.81) and H-6 (δ 4.32), but there was no NOE observed between H-3 (δ 5.37) and H-6. Examination of molecular models revealed the H-5 and H-6 can show an NOE regardless of whether they are on the same or opposite sides of the oxetane ring, but the absence of a H-3/H6 NOE indicated that C-3 and H-6 must be on opposite faces, as shown in the 4S*,5S*,6S* relative configuration assigned in 2. We have assumed that the absolute configurations at C-6 and C-5 are identical in ramariolides A (1) and B (2). This assumption, which is consistent with the syn C-3/H-5 and anti C-3/H-6 relative configurations assigned above, gives the 4S,5S,6S absolute configuration for 2 as shown. Ramariolide C (3) was isolated as an optically active, white solid that gave a [M − H]− ion in the HRESIMS at m/z 265.1802, appropriate for a molecular formula of C16H26O3, requiring four sites of unsaturation. Extensive analysis of the 1D and 2D NMR data revealed many of the same structural features seen in 1, with some intriguing differences. The lactone (C-1, δ 168.9) with α,β-unsaturation (C-2, δ 121.3 and C-3, δ 140.4) was present, but the UV λmax at 273 nm was higher than observed for 1 and 2. HMBC correlations observed between both H-2 (δ 5.54) and H-3 (δ 6.88) and an olefinic carbon resonance at δ 150.6, assigned to C-4, and between H-3 and a resonance at δ 117.0, assigned to C-5, identified a Δ4,5 alkene. The extended conjugation agreed with the bathochromic shift in the UV data. COSY correlations were observed between an oxymethine resonance at δ 3.81 (H-6) and H-5 (δ 5.29), a doublet at δ 0.69 (with no carbon correlation in the gHSQC), and a pair of diastereotopic methylene proton resonances at δ 1.30 (H-7) and δ 1.23 (H-7′). These COSY correlations identified a secondary alcohol at C-6 and an aliphatic methylene at C-7 as in 1 and 2. The remainder of the NMR data were consistent with a saturated linear 10-carbon alkyl chain extending from C-7 to C-16. A ROESY correlation between H-6 (δ 3.81) and H-3 (δ 6.88) showed that the Δ4,5 alkene had the E configuration. We have assumed that C-6 in 3 has the S configuration as in ramariolide A (1). Ramariolide D (4) was isolated as an optically active, white solid that gave a [M + H]+ ion in the HRESIMS at m/z 287.1633, appropriate for a molecular formula of C16H24O3 (calcd 287.1623) requiring five sites of unsaturation. Analysis of the NMR data obtained for 4 showed that it differed from 3 only in the alkyl side chain. Two olefinic resonances in the 13C NMR spectrum at δ 140.4 (C-15) and δ 115.0 (C-16), which showed HSQC correlations to an olefinic methine at δ 5.81 (H15) and two geminal olefinic doublets at δ 5.07 and δ 5.01 (H16/H-16′), respectively, identified a terminal Δ15,16 alkene on
Figure 4. Antimycobacterial activity of ramariolide A (1). (A) Resazurin assay for activity against M. smegmatis. Pink/purple indicates live cells, blue indicates dead cells. Twofold serial dilutions were performed from top to bottom. The highest concentration tested for 1 was 128 μg/mL. Isoniazid (INH) and spectinomycin (SPT) were included for comparison at maximum concentrations of 64 μg/mL. Control wells contained untreated cells (CT+) or only media (CT−). (B) MTT assay for activity against M. tuberculosis. A log2(dose)− response nonlinear (four parameters) fitting curve was used to calculate the IC50. The 95% confidence interval of the IC50 was 5.161 to 6.329 (35.77 to 80.39 μg/mL). Experiments were performed in triplicate.
antituberculosis bactericidal antibiotic, had an MIC of 8 μg/mL and a MBC/MIC ratio of 1; spectinomycin, a bacteriostatic agent not used therapeutically for treatment of tuberculosis, had an MIC of 32 μg/mL and a MBC/MIC ratio of 2−4. Ramariolide A (1) was active against several strains of M. tuberculosis; the concentration needed for 50% inhibition of growth (IC50) was 53 μg/mL and the MIC was 64−128 μg/mL (Figure 4B). In addition to its antimycobacterial effect, ramariolide A (1) also inhibited growth of Micrococcus luteus and Staphylococus aureus (inhibition zones of 18 and 15 mm 2180
dx.doi.org/10.1021/np3006277 | J. Nat. Prod. 2012, 75, 2178−2182
Journal of Natural Products
Article
times: 1−27.5, 2−26, 3−25.5, and 4−19 min), yielding 1 (10 mg), 2 (0.5 mg), 3 (0.3 mg), and 4 (0.1 mg). Ramariolide A (1): irregular, colorless crystals (CH2Cl2); mp 92− 94 °C; [α]25D −42 (c 5.4, MeOH); UV (MeOH) λmax (log ε) 213 (3.3) nm; 1H and 13C NMR see Table 1; HRESIMS [M − H]− m/z 281.1769 (calcd for C16H25O4, 281.1753).
diameter, respectively, on disk diffusion assays), but there was no effect on Gram-negative tester strains (see Experimental Section). Ramariolides A−D (1−4) represent a novel carbon chain length for unbranched butenolides. The most similar compounds to the ramariolides in the literature are the hygrophorones F and G,10 which resemble ramariolide C (3) in structure but contain two additional methylenes in the alkyl chain. The spiro oxiranebutenolide and spiro oxetanebutenolide moieties in 1 and 2 represent novel functionalities for polyketide-derived natural products, and the spirooxetanebutenolide in 2 is only the second time this functionality has been observed in nature, the other example being in the sesquiterpenoid parthexetine.11 R. cystidiophora is represented by several varieties in the field, and it would be interesting to determine if the ramariolides are a varietal characteristic or distributed more widely in other Ramaria species or taxa. The results of this study suggest this genus merits further investigation as a source of novel compounds with antimicrobial potential.
■
Table 1. NMR Data for Ramariolide A (1) and Ramariolide B (2) ramariolide A (1)a position 1 2 3 4 5 6 7 8 9 to 13
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured using a Jasco P-1010 polarimeter with sodium light (589 nm). UV spectra were recorded with a Waters 2487 Dual λ absorbance detector. The 1H and 13C NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5 mm CPTCI cryoprobe. 1H chemical shifts are referenced to the residual benzene-d6, dichloromethane-d2, or chloroform-d3 signal (δ 7.15, 5.32, 7.26 ppm, respectively), and 13C chemical shifts are referenced to the benzene-d6 or dichloromethane-d2 solvent peak (δ 128.0 or 53.8 ppm, respectively). Low- and highresolution ESI-QIT-MS were recorded on a Bruker-Hewlett-Packard 1100 Esquire-LC system mass spectrometer. Merck type 5554 silica gel plates and Whatman MKC18F plates were used for analytical thinlayer chromatography. Reversed-phase HPLC purifications were performed on a Waters 1525 Binary HPLC pump attached to a Waters 2487 Dual λ absorbance detector. All solvents used for HPLC were Fisher HPLC grade. Mushroom Sample. The source specimen, W179, was a display sample used in the 2009 Vancouver Mycological Society Fall Mushroom show, in which members collected examples of local (southwestern British Columbia) fungi for exhibition to the public. The sample was donated for the purpose of this study and identified specifically as Ramaria cytidiophora using morphological4 and genetic criteria. For the latter, DNA was extracted using standard procedures, and the ITS12 region between the 18S and 28S rRNA genes was amplified by PCR and sequenced. When compared to publicly available accessions in GenBank, the 511 nucleotides obtained were observed to be identical to accessions EU597077 (nt 34-544) and DQ384590 (nt 79-589), both derived from samples originating in southwestern British Columbia and previously identified as R. cystidiophora. A voucher sample of W179 has been deposited in the herbarium at the University of British Columbia. Extraction and Isolation. Approximately 110 g fresh weight of mushroom was cut into pieces and extracted three times with MeOH. The combined extracts were concentrated in vacuo at rt to yield an orange solid, which was partitioned between 200 mL of H2O and EtOAc. The EtOAc was concentrated in vacuo to give 0.27 g of residue, which was then chromatographed on Sephadex LH20 (3 cm × 95 cm), yielding four fractions. The fourth fraction, containing the compounds of interest (monitored by TLC), was subjected to C18 reversed-phase HPLC using a CSC-Inertsil 150 Å/ODS2, 5 μm, 25 × 0.94 cm column, with 4:5 MeCN/H2O as eluent to yield all four compounds in a nearly pure state (retention times of 1−19.5, 2−17, 3−18.5, and 4−14 min). Final purification was done using C8 reversed-phase HPLC using a Phenomenex Luna C8100 Å, 5 μm 250 × 10 mm, column with 7:10 MeCN/H2O as eluent (retention
14 15 16 OH-6 OH-5
δC, type 168.9, 126.7, 149.7, 90.4, 65.5, 67.9, 34.1, 25.6, 30.1, 30.1, 30.0, 30.0, 29.9, 32.4, 23.2, 14.4,
δH (J in Hz)
C CH CH C CH CH CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3
6.34, d (5.6) 7.55, d (5.6) 3.77, 4.05, 1.62, 1.37, 1.27,
d (4.1) m 1.49, m bm bm
1.25, 1.27, 0.88, 1.26,
m m t (7.2) bm
ramariolide B (2)b δC 168.0 124.5 148.8 112.7 74.5 90.7 34.9 24.9 29.9, 29.8, 29.8, 29.6, 29.5
23.0 32.2 14.9
δH (J in Hz) 5.37, d (5.6) 6.06, d (5.6) 3.81, dd (6.1) 4.33, q (6.7) 1.47, 1.38, bm 1.18, bm 1.5, bm
1.29, bm 1.28, bm 0.93, t (7.2) 2.12, d (12.3)
a
Recorded in methylene chloride-d2 (1H 600 MHz, 13C 150 MHz). b Recorded in benzene-d6 (1H 600 MHz, 13C 150 MHz). Ramariolide B (2): clear oil; [α]25D −50 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 203 (3.2) nm; 1H and 13C NMR see Table 1; HRESI [M + Na]+ m/z 305.1721 (calcd for C16H26O4Na, 305.1729). Ramariolide C (3): clear, amorphous oil; [α]25D +85 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 273 (3.1) nm; 1H and 13C NMR see Table 2; HRMS (ESI) [M − H]− m/z 265.1802 (calcd for C16H25O3, 265.1804). Ramariolide D (4): clear, amorphous solid; [α]25D +42 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 273 (3.4) nm; 1H and 13C NMR see Table 2; HRMS (ESI) [M + H]+ m/z 287.1633 (calcd for C16H24O3Na, 287.1623). Bacterial Strains and Growth Inhibition Assays. Gramnegative bacteria (Escherichia coli O157:H7, Acinetobacter baumanii ATCC 19606, Pseudomonas aeruginosa PAO1 H103, Salmonella typhimurium ATCC 14028), Gram-positive bacteria (S. aureus RN4220, M. luteus JVC 1154), and M. smegmatis mc2155 were grown and assayed at 37 °C in NE13 medium; M. tuberculosis strains (H37Rv, CDC1551, and Erdman) were grown in Middlebrook 7H9 broth (Difco) supplemented with 0.2% glycerol, 10% Middlebrook ADC (Difco), and 0.05% (v/v) Tyloxapol. Tyloxapol was not incorporated in media for assays (below). The MIC against M. smegmatis was determined in triplicate using a colorimetric redox indicator, resazurin, in broth assays.14 The MIC was defined as the lowest concentration of compound that prevented a color change in the medium from blue (no cellular activity) to pink (metabolically active cells). To determine the MBC, a 5 μL aliquot was transferred from each well into 200 μL of fresh medium before adding resazurin to the remaining culture for the MIC assay; the subculture was processed as described for MIC determinations. Isoniazid and spectinomycin (Sigma) were used as comparators. An MBC/MIC ratio > 2 indicates bacteriostatic activity; a ratio ≤ 2 indicates cidality.14 The MIC against M. tuberculosis strains (H37Rv, CDC1551, and Erdman) was conducted using the MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide] assay.15 In brief, microtiter plates 2181
dx.doi.org/10.1021/np3006277 | J. Nat. Prod. 2012, 75, 2178−2182
Journal of Natural Products
Article
Table 2. NMR Data (benzene-d6, 1H 600 MHz, 13C 150 MHz) for Ramariolides C (3) and D (4) ramariolide C (3) position
δC, type
1 2
168.9, C 121.3, CH
3 4 5
140.4, CH 150.6, C 117.0, CH
6 7 8 9 to 13
13 14 15 16
68.2, 38.2, 25.7, 30.4,
CH2 CH2 CH2 CH2
30.4, 30.3, 30.2, 30.1,
CH2 CH2 CH2 CH2
32.5, CH2 23.5, CH2 14.7, CH3
OH-6
δH (J in Hz) 5.54, dd (1.6, 4.0) 6.88, d (5.6) 5.29, d (0.8, 7.2) 3.81, 1.30, 1.03, 1.55,
bm 1.23, bm bm bm
1.29, bm 1.38, bm 0.93, t (6.7) 0.69, d (4.0)
of Health Research (grant MOP-82855 to C.J.T.) and NSERC (to R.J.A.).
■
ramariolide D (4) δC, type 167.9, C 120.2, CH 139.3, CH 149.8, C 115.7, CH 67.2, CH2 37.4, CH2 24.8, CH2 29.2, CH2 29.1, CH2 29.1, CH2 28.8, CH2
28.6, 33.3, 138.4, 113.7,
CH2 CH2 CH CH2
δH (J in Hz)
(1) World Health Organization. Global Tuberculosis Control; World Health Organization: Geneva, Switzerland. 2011. (2) García, A.; Bocanegra-García, V.; Palma-Nicolás, J. P.; Rivera, G. Eur. J. Med. Chem. 2012, 49, 1−23. (3) Roemer, T.; Xu, D.; Singh, S. B.; Parish, C. A.; Harris, G.; Wang, H.; Davies, J. E.; Bills, G. F. Chem. Biol. 2011, 18, 148−64. (4) Exeter, R. L.; Norvell, L.; Cázares, E. Ramaria of the Pacific Northwestern United States; United States Department of the Interior, Bureau of Land Management: Salem, OR. 2006. (5) Dunham, S. M.; Larsson, K. H.; Spatafora, J. W. Mycorrhiza 2007, 17, 633−645. (6) Humpert, A.; Muench, E.; Giachini, A.; Castellano, M.; Spatofora, J. Mycologia 2001, 93, 465−477. (7) Yaoita, Y.; Satoh, Y.; Kikuchi, M. J. Nat. Med. 2007, 61, 205−207. (8) Steglich, W.; Steffan, B.; Stroech, K.; Wolf, M. Z. Naturforsch. 1984, 39, 10−12. (9) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092−4096. (10) Lübken, T.; Schmidt, J.; Porzel, A.; Arnolde, N.; Wessjohann, L. Phytochemistry 2004, 65, 1061−1071. (11) Ortega, A.; Maldonado, E. Phytochemistry 1986, 25, 699−701. (12) Nouhra, E. R.; Horton, T. R.; Cazares, E.; Castellano, M. Mycorrhiza 2005, 15, 55−59. (13) Murakami, T.; Anzai, H.; Imai, S.; Satoh, A.; Nagoaka, K.; Thompson, C. J. Mol. Gen. Genet. 1986, 205, 42−50. (14) Ramon-Garcia, S.; Ng, C.; Anderson, H.; Chao, J. D.; Zheng, X.; Pfeifer, T.; Av-Gay, Y.; Roberge, M.; Thompson, C. J. Antimicrob. Agents Chemother. 2011, 55, 3861−3869. (15) Montoro, E.; Lemus, D.; Echemendia, M.; Martin, A.; Protaels, F.; Palomino, J. C. J. Antimicrob. Chemother. 2005, 55, 500−505.
5.54, dd (2.1, 3.6) 6.88, d (5.6) 5.29, dd (1.5, 6.7) 3.81, q (5.1) 1.28, 1.14, bm 1.01, bm 1.45, bm
1.35, 2.02, 5.81, 5.02, 5.08, 0.68,
bm q (7.2) m d (5.0) d (10.0) bm
with wells containing 100 μL of inoculum (105 cells/mL) and 2-fold serial dilutions of ramariolide A (1) were incubated at 37 °C, in 5% CO2. After 7 d, 50 μL of MTT reagent (2.5 mg/mL in H2O) was added to each well, and the plates were reincubated for 24 h. Then, 100 μL of 10% Na lauryl sulfate was added per well to solubilize the formazan precipitate formed in the presence of metabolically active cells, and the OD570 was measured after 24 h at 37 °C. Disk diffusion assays were used for acitivity spectrum analysis: paper disks (6 mm diameter, BBL) with 50 μg of ramariolide A (1) were placed on agar plates swabbed from suspensions (106 cells/mL) of each tested strain, and zones of growth inhibition were measured after 16−18 h.
■
REFERENCES
ASSOCIATED CONTENT
S Supporting Information *
1D and 2D NMR spectra are included for ramariolides A to D (1−4) along with details of the X-ray diffraction and Mosher ester analysis of 1. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Tel: 604-822-4511. Fax: 604-822-2847. E-mail: randersn@ mail.ubc.ca. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We are grateful to the Vancouver Mycological Society for donating material for this study, to L. Fei for technical assistance, and to Dr. J. Davies at the University of British Columbia for allowing some of the work to be done in his laboratory. Financial support was provided by grants from the British Columbia Lung Association and the Canadian Institute 2182
dx.doi.org/10.1021/np3006277 | J. Nat. Prod. 2012, 75, 2178−2182
