Environment Health Techniques S70
Dong-Ze Liu et al.
Short Communication Two new alkaloids from the edible macrofungus Ramaria madagascariensis Dong-Ze Liu1, Jian-Guo Li2, Ming-Wei Zhang2 and Gang Liu2 1 2
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China Zhejiang University, Hangzhou, China
Phytochemical investigation of the ethyl acetate extract of the edible macrofungus, Ramaria madagascariensis, has led to the isolation of two new alkaloids (1 and 2). Their structures were elucidated by HR-ESI-MS, IR, and 1D- and 2D-NMR experiments. The antimicrobial activity of 1 and 2 was also tested and evaluated. Keywords: Alkaloids / Isolation / Ramaria madagascariensis / Fruiting Bodies / 2D-NMR Received: December 30, 2013; accepted: March 20, 2014 DOI 10.1002/jobm.201301060
Introduction
Materials and methods
Mushrooms have attracted much more attention as functional foods or physiologically beneficial medicine due to their nutritional and medicinal value and the diversity of their bioactive secondary metabolites [1, 2]. In recent years, these fungi have been reported to possess a variety of bioactivities, such as antitumor, antimicrobial, hypoglycemic, and antioxidant [3–5]. Ramaria madagascariensis, a wild edible mushroom, which belongs to Ramariacea family, is mainly distributed in Jilin Province, Yunnan Province, and the Tibet Autonomous Region of China. This wild mushroom has traditionally been eaten by specific groups of local people and seasonally in China. However, little is known about the chemical composition and nutritional and pharmacological values of this mushroom. In our continuous screening for naturally occurring bioactive metabolites [6–9], chemical studies of the fruiting bodies of R. madagascariensis led to the isolation and characterization of two new alkaloids (1 and 2). Their structures (Fig. 1) were elucidated by HR-ESI-MS, IR, and 1D- and 2D-NMR experiments. In addition, the antimicrobial activity of these two compounds was tested and evaluated.
General experimental procedures Infrared (IR) spectra were obtained on a Bruker Tensor 27 spectrometer (Bruker Optics, Ettlingen, Germany) with KBr pellets. Ultraviolet (UV) spectra were obtained on a UV–vis Evolution 300 spectrometer (Thermo Scientific, USA). Optical rotation was measured on a Horiba SEPA300 polarimeter (Horiba, Kyoto, Japan). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 500 MHz spectrometer (Bruker BioSpin, Rheinstetten, German) with tetramethylsilane (TMS) as internal standard. Electrospray ionization mass spectrometry (ESIMS) data were obtained on a Thermo Fisher LTQ Fleet instrument spectrometer (Thermo Scientific). High-resolution electrospray ionization mass spectrometry (HRESIMS) data were recorded on an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies, USA). Column chromatography (CC) was performed using silica gel (200–300 mesh; Qingdao Marine Chemicals, Qingdao, China) or Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). Semipreparative HPLC was performed on an Agilent 1200 HPLC system using an ODS column (RP-8, 250 mm 10 mm, YMC Pak, 5 mm; detector: UV) with a flow rate of 2.0 ml min1. Fractions were monitored by TLC (GF254, Qingdao Marine Chemical Co., Ltd., Qingdao, China), and spots were visualized by spraying with 5% H2SO4 in ethanol, followed by heating.
Correspondence: Dong-Ze Liu, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China E-mail: [email protected] Phone/Fax: þ86 22 84861942 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
www.jbm-journal.com
J. Basic Microbiol. 2014, 54, S70–S73
New alkaloids from Ramaria madagascariensis
Figure 1. The structures of compounds 1 and 2.
Plant materials The fruiting bodies of R. madagascariensis (collected in Tibet, China) were supplied and identified by one of the authors (G. Liu). The voucher specimen (201004281) was deposited in the college of Agriculture and Biotechnology, Zhejiang University, China. Extraction and isolation The air-dried and powdered fruit bodies of R. madagascariensis (2.5 kg) were extracted with 95% EtOH (10 L 3) at room temperature for 6 days. The EtOH extract was next partitioned between EtOAc and H2O with the organic layer evaporated in vacuo to afford the EtOAc extract (45 g). The latter was subjected to a silica gel column chromatography eluted with increasing polarities of a mixture of CHCl3 and MeOH, and seven fractions (Fr.1Fr.7) were obtained on the basis of TLC analysis. Fraction 3 was separated on a silica gel column eluted with petroleum ether/EtOAc (10:1 to 0:1 v/v) to give five subfractions (Fr.3-1–Fr.3-5). Fr.3-3 was rechromatographed on Sephadex LH-20 (CHCl3/MeOH, 1:1 v/v) followed by semipreparative reversed-phase HPLC (30% CH3CN in H2O over 10 min, 30–75% over 50 min) to afford compound 2 (3.5 mg, tR 21.5 min). Fr. 3-4 was seperated repeatedly by silica gel eluting with petroleum ether/EtOAc (8:1–3:1 v/v) to give compound 1 (10.2 mg). Antibacterial assay Compounds 1 and 2 were tested for antibacterial activity using a disk diffusion assay [10]. Seed cultures of three bacteria, Staphylococcus aureus ATCC29213, Bacillus thuringiensis ATCC39765, and Bacillus subtilis ATCC6633, were cultivated in LB broth medium by incubating the organisms for 10 h at 37 °C. Then the seed cultures were added to LB agar medium to make plates. Sterile filter disks (6 mm diameter) infused with 5 mL of test solution ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
S71
(2 mg mL1 DMSO) and positive control (5 mg mL1 DMSO ampicillin) were added to the plates. The plates were placed in an incubator at 37 °C. After 12 h, the diameters of the zones of growth inhibition around each disk were recorded. (1) white powder; m.p. 159–158 °C; ½a25 D 24:1 (c 0.07, MeOH), UV (CH3OH) lmax (log e): 214 (4.50) nm; IR (KBr) nmax: 3447, 3289, 2926, 1740, 1685, 1643, 1537, 975 cm1; 1 H and 13C NMR spectroscopic data (see Table 1); (þ)-HRESIMS m/z 356.1835 [MþNa]þ (calcd. for C18H27NO6Na, 356.1838). (2) Yellow powder; m.p. 131–132 °C; ½a25 D 36:3 (c 0.08, MeOH), UV (CH3OH) lmax (log e): 215(4.12) nm; IR (KBr) nmax: 3391, 3302, 2926, 1689, 1669, 1630, 1551, 1036 cm1; 1H and 13C NMR spectroscopic data (see Table 1); (þ)-HRESIMS m/z 402.1889 [MþNa]þ (calcd. for C20H29NO6Na, 402.1893).
Results and discussion Compound 1 was isolated as a colorless powder. The molecular formula was determined by HRESIMS as C18H27NO6, which suggested 6° of unsaturation. The 1 H, 13C, and DEPT NMR spectra indicated the presence of two methyl (oxygenated), five methylenes, eight methines (two olefinic and four oxygenated), and three quaternary carbons (a ketone carbonyl, an amide carbonyl, and one oxygenated). Accordingly, a bicyclic structure was required for 1 to fulfill the unsaturation requirement. The 1H–1H COSY spectrum (Fig. 2) revealed four 1H–1H spin systems of H-5/H-6/H-7/H-8, H-1/H-2/H-3, H-2/NH, and H-1/OH. The aforementioned data, along with key HMBC correlations (Fig. 2) originated from H-1, H2-3, OH-4, H-5, H-7, and H-8, led to the determination of the bridged cyclic structure. The location of the methoxy group was indicated by the HMBC correlations from OCH3 to C-7, and H-7 to 7-OCH3. The carboxyamide side chain was inferred upon detailed 1H–1H COSY and HMBC spectroscopic analysis. The HMBC correlation of H-2 to C-10 located the carboxyamide side chain at C-2. Taking the molecular formula and the degrees of unsaturation as well as the observation of corresponding resonances for CH-5 and CH-6, the presence of a cis-epoxide was suggested in the structure. By combining all this evidence and data, we were able to assign the gross structure of 1. The geometry of C-20 /C-30 double bond was determined to be E, on the basis of NOESY correlations of H-20 to H-40 . The relative configuration of 1 was deduced from NOESY spectroscopic data and 1H NMR coupling constants. The NOESY cross-peak between H-5 and H-6 and a coupling constant of 3.7 Hz indicated the cis-epoxide ring. The
www.jbm-journal.com
J. Basic Microbiol. 2014, 54, S70–S73
S72
Dong-Ze Liu et al.
Table 1. 1H (500 MHz) and
13
C (125 MHz) NMR Data for 1 and 2 (acetone-d6). 1
No.
dH
1 2 3
4.38 4.74 2.30 2.01
(m) (m) (ddd,12.6,5.0,1.7) (t,12.6)
3.53 3.45 3.87 3.06
(dd,3.7,1.2) (dd,3.7,1.5) (dd,7.5,1.2) (ddd,7.5,3.4,1.5)
6.04 6.74 2.15 1.44 1.29 1.28 0.86
(dt,15.1, 1.5) (dt,15.1, 7.0) (m) (m) (m) (m) (t, 7.0)
4.62 4.48 3.48 7.97
(d, 3.6) (s) (s) (br d, 8.1)
4 5 6 7 8 9 10 20 30 4 50 60 70 80 90 100 OH-1 OH-4 OCH3 NH
2 dC 71.3 46.3 38.1 75.7 63.8 55.4 77.5 55.1 207.3 164.8 124.2 143.8 31.6 28.3 31.8 22.4 13.6
57.5
large coupling constant (12.6 Hz) suggested a trans-diaxial orientation of H-2 and H-3b. A W-type long-range coupling (J ¼ 1.7 Hz) was observed between two equatorial protons, H-1 and H-3a. Irradiation of H-7 in a NOEDIFF experiment resulted in signal enhancement of H-1, while irradiation of H-3a gave signal enhancement of H-5. From the above analysis, the structure of 1 was established as shown.
Figure 2. Key COSY and HMBC correlations in 1 and 2. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
dH
dC
5.20 4.52 1.98 1.81
(dd,4.4, 3.8) (m) (dd,13.5,4.5) (t,13.5)
4.09 4.26 6.90 5.89
(d,8.3) (dt,8.3,2.1) (dd,10.5,2.1) (dd, 10.5, 2.1)
6.05 6.74 2.16 1.55 1.32 1.98 5.43 5.43 1.60 5.81 4.93 3.47 6.88
(dt,15.1, 1.5) (dt,15.1, 7.0) (m) (m) (m) (m) (m) (m) (m) (br d, 4.4) (s) (s) (br d, 8.9)
90.2 44.8 30.1 72.7 73.5 76.3 148.4 126.3 196.2 164.7 124.3 143.6 31.5 27.7 28.7 32.3 131.8 124.1 17.2 57.7
Compound 2 was obtained as yellow amorphous powder. Its molecular formula was established as C20H29NO6 by HRESIMS data (m/z 402.1889 [MþNa]þ, calcd. 402.1893), which suggested 7° of unsaturation. The IR spectrum showed absorption bands at 3380 (OH), 1689 (conjugated ketone), and 1669 (amide carbonyl) cm1. The 1H, 13C, and DEPT NMR spectra indicated the presence of 2 methyl (oxygenated), 5 methylenes, 10 methines (6 olefinic, a hemiacetal, and 2 oxygenated), and 3 quaternary carbons (a ketone carbonyl, an amide carbonyl, and 1 oxygenated). Accordingly, a bicyclic structure was required for 2 to fulfill the unsaturation requirement. The 1H–1H COSY spectrum (Fig. 2) revealed four 1H–1H spin systems of H-5/H-6/H-7/H-8, H-1/H-2/H-3, H-2/NH, and H-1/OH. The aforementioned data, along with key HMBC correlations (Fig. 2) originated from H-1, H2-3, OH-4, H-5, H-7, and H-9, led to the determination of the bicyclic core. The location of the methoxy group was indicated by the HMBC correlations from OCH3 to C-6, and H-6 to 6-OCH3. The carboxyamide side chain was inferred upon detailed 1H–1H COSY and HMBC spectroscopic analysis. The HMBC correlation of H-2 to C-10 located the carboxyamide side chain at C-2. By combining all this evidence and data, we were able to assign the gross structure of 2. The geometries of the C-7/C-8 and C20 /C-30 double bonds were determined to be Z and E, respectively, on the basis of NOESY correlations of H-7 to
www.jbm-journal.com
J. Basic Microbiol. 2014, 54, S70–S73
New alkaloids from Ramaria madagascariensis
H-8 and H-20 to H-40 . Due to the overlap of the olefinic proton resonances, trans configuration (80 E) was addressed by comparison of the chemical shifts of C-70 C-100 with corresponding carbons for trans-2-heptene (C1 dC 17.9, C-2 dC 124.6, C-3 dC 131.7, C-4 dC 32.4) and cis-2heptene (C-1 dC 12.7, C-2 dC 123.7, C-3 dC 130.9, C-4 dC 26.7). The relative configuration of 2 was deduced on the basis of 1H–1H J values and NOESY spectroscopic data. A NOESY cross-peak observed between H-3b and H-5 and the large coupling constant, J2,3b of 13.5 Hz, indicated the axial orientations of H-2, H-3b, and H-5, and trans ring junction, while the 1H–1H J value, J1,2 of 3.8 Hz, suggested an equatorial position of H-1. The large coupling constant for H-5 and H-6 (J ¼ 8.3 Hz) suggested b-position of H-5 and a-position of H-6. From the above evidence, the structure of 2 was established as shown. Structurally, compounds 1 and 2 possess chemical skeletons similar to isariotins E-J and TK-57-164A [10–14] except for the carboxyamide side chain or chloro substituent on the cyclic core. Accordingly, biosynthetic routes to compounds 1 and 2 may resemble those for the above compounds starting with condensation of the corresponding unsaturated carboxylic acid and L-tyrosine [14]. Compounds 1 and 2 were evaluated for their antimicrobial activity. However, none of them showed an inhibition zone at a concentration of 100 mg/disk.
References [1] Barros, L., Calhelha, R.C., Vaz, J.A., Ferreira, I.C.F. et al., 2007. Antimicrobial activity and bioactive compounds of Portuguese wild edible mushrooms. Eur. Food. Res. Technol., 225, 151–156. [2] Barro, L., Baptista, P., Estevinho, L.M., Ferreira, I.C.F., 2007. Bioactive properties of the medicinal mushroom Leucopaxillus giganteus mycelium obtained in the presence of different nitrogen sources. Food Chem., 105, 179–186. [3] Li, S.P., Zhang, G.H., Zeng, Q., Huang, Z.G. et al., 2006. Hypoglycemic activity of polysaccharide, with antioxida-
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
S73
tion, isolated from cultured Cordyceps mycelia. Phytomedicine, 13, 428–433. [4] Xiao, J.H., Xiao, D.M., Chen, D.X., Xiao, Y. et al., 2012. Polysaccharides from the medicinal mushroom Cordyceps taii show antioxidant and immunoenhancing activities in a D-galactose-induced aging mouse model. Evid.-Based Complement. Altern. Med., 2012, 273–435. [5] Xu, T., Beelman, R.B., Lambert, J.D., 2012. The cancer preventive effects of edible mushrooms. Anticancer Agents Med. Chem., 12, 1255–1263. [6] Liu, D.Z., Dong, Z.J., Wang, F., Liu, J.K., 2010. Two novel norsesquiterpene peroxides from basidiomycete Steccherinum ochraceum. Tetrahedron Lett., 51, 3152–3153. [7] Liu, D.Z., Wang, F., Liao, T.G., Tang, J.G. et al., 2006. Vibralactone: a lipase inhibitor with an unusual fused bLactone produced by cultures of the basidiomycete Boreostereum vibrans. Org. Lett., 25, 5749–5752. [8] Liu, D.Z., Wang, F., Liu, J.K., 2007. Sesquiterpenes from cultures of the basidiomycete Conocybe siliginea. J. Nat. Prod., 70, 1503–1506. [9] Liu, D.Z., Jia, R.R., Wang, F., Liu, J.K., 2008. A new spiroaxane sesquiterpene from cultures of the basidiomycete Pholiota adiposa. Z. Naturforsch., 63b, 111–113. [10] Wang, X., Filho, J.G.S., Hoover, A.R., King, J.B. et al., 2010. Chemical epigenetics alters the secondary metabolite composition of guttate excreted by an Atlantic-forestsoil-derived Penicillium citreonigrum. J. Nat. Prod. 73, 942–948. [11] Asai, T., Chung, Y.M., Sakurai, H., Ozeki, T. et al., 2012. Highly oxidized ergosterols and isariotin analogs from an entomopathogenic fungus, Gibellula formosana, cultivated in the presence of epigenetic modifying agents. Tetrahedron, 68, 5817–5823. [12] Bunyapaiboonsri, T., Yoiprommarat, S., Srisanoh, U., Choowong, W. et al., 2011. Isariotins G–J from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes. Phytochem. Lett., 4, 283–286. [13] Cha, J.Y., Huang, Y.D., Pettus, T.R.R., 2009. Total synthesis of TK-57-164A, isariotin F, and their putative progenitor isariotin E. Angew. Chem. Int. Ed., 48, 9519–9521. [14] Bunyapaiboonsri, T., Yoiprommarat, S., Intereya, K., Rachtawee, P. et al., 2009. Isariotins E and F, spirocyclic and bicyclic hemiacetals from the entomopathogenic fungus Isaria tenuipes BCC 12625. J. Nat. Prod., 72, 756–759.
www.jbm-journal.com
J. Basic Microbiol. 2014, 54, S70–S73
Dong-Ze Liu et al.
Short Communication Two new alkaloids from the edible macrofungus Ramaria madagascariensis Dong-Ze Liu1, Jian-Guo Li2, Ming-Wei Zhang2 and Gang Liu2 1 2
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China Zhejiang University, Hangzhou, China
Phytochemical investigation of the ethyl acetate extract of the edible macrofungus, Ramaria madagascariensis, has led to the isolation of two new alkaloids (1 and 2). Their structures were elucidated by HR-ESI-MS, IR, and 1D- and 2D-NMR experiments. The antimicrobial activity of 1 and 2 was also tested and evaluated. Keywords: Alkaloids / Isolation / Ramaria madagascariensis / Fruiting Bodies / 2D-NMR Received: December 30, 2013; accepted: March 20, 2014 DOI 10.1002/jobm.201301060
Introduction
Materials and methods
Mushrooms have attracted much more attention as functional foods or physiologically beneficial medicine due to their nutritional and medicinal value and the diversity of their bioactive secondary metabolites [1, 2]. In recent years, these fungi have been reported to possess a variety of bioactivities, such as antitumor, antimicrobial, hypoglycemic, and antioxidant [3–5]. Ramaria madagascariensis, a wild edible mushroom, which belongs to Ramariacea family, is mainly distributed in Jilin Province, Yunnan Province, and the Tibet Autonomous Region of China. This wild mushroom has traditionally been eaten by specific groups of local people and seasonally in China. However, little is known about the chemical composition and nutritional and pharmacological values of this mushroom. In our continuous screening for naturally occurring bioactive metabolites [6–9], chemical studies of the fruiting bodies of R. madagascariensis led to the isolation and characterization of two new alkaloids (1 and 2). Their structures (Fig. 1) were elucidated by HR-ESI-MS, IR, and 1D- and 2D-NMR experiments. In addition, the antimicrobial activity of these two compounds was tested and evaluated.
General experimental procedures Infrared (IR) spectra were obtained on a Bruker Tensor 27 spectrometer (Bruker Optics, Ettlingen, Germany) with KBr pellets. Ultraviolet (UV) spectra were obtained on a UV–vis Evolution 300 spectrometer (Thermo Scientific, USA). Optical rotation was measured on a Horiba SEPA300 polarimeter (Horiba, Kyoto, Japan). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III 500 MHz spectrometer (Bruker BioSpin, Rheinstetten, German) with tetramethylsilane (TMS) as internal standard. Electrospray ionization mass spectrometry (ESIMS) data were obtained on a Thermo Fisher LTQ Fleet instrument spectrometer (Thermo Scientific). High-resolution electrospray ionization mass spectrometry (HRESIMS) data were recorded on an Agilent 6520 Accurate-Mass Q-TOF LC/MS spectrometer (Agilent Technologies, USA). Column chromatography (CC) was performed using silica gel (200–300 mesh; Qingdao Marine Chemicals, Qingdao, China) or Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). Semipreparative HPLC was performed on an Agilent 1200 HPLC system using an ODS column (RP-8, 250 mm 10 mm, YMC Pak, 5 mm; detector: UV) with a flow rate of 2.0 ml min1. Fractions were monitored by TLC (GF254, Qingdao Marine Chemical Co., Ltd., Qingdao, China), and spots were visualized by spraying with 5% H2SO4 in ethanol, followed by heating.
Correspondence: Dong-Ze Liu, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China E-mail: [email protected] Phone/Fax: þ86 22 84861942 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
www.jbm-journal.com
J. Basic Microbiol. 2014, 54, S70–S73
New alkaloids from Ramaria madagascariensis
Figure 1. The structures of compounds 1 and 2.
Plant materials The fruiting bodies of R. madagascariensis (collected in Tibet, China) were supplied and identified by one of the authors (G. Liu). The voucher specimen (201004281) was deposited in the college of Agriculture and Biotechnology, Zhejiang University, China. Extraction and isolation The air-dried and powdered fruit bodies of R. madagascariensis (2.5 kg) were extracted with 95% EtOH (10 L 3) at room temperature for 6 days. The EtOH extract was next partitioned between EtOAc and H2O with the organic layer evaporated in vacuo to afford the EtOAc extract (45 g). The latter was subjected to a silica gel column chromatography eluted with increasing polarities of a mixture of CHCl3 and MeOH, and seven fractions (Fr.1Fr.7) were obtained on the basis of TLC analysis. Fraction 3 was separated on a silica gel column eluted with petroleum ether/EtOAc (10:1 to 0:1 v/v) to give five subfractions (Fr.3-1–Fr.3-5). Fr.3-3 was rechromatographed on Sephadex LH-20 (CHCl3/MeOH, 1:1 v/v) followed by semipreparative reversed-phase HPLC (30% CH3CN in H2O over 10 min, 30–75% over 50 min) to afford compound 2 (3.5 mg, tR 21.5 min). Fr. 3-4 was seperated repeatedly by silica gel eluting with petroleum ether/EtOAc (8:1–3:1 v/v) to give compound 1 (10.2 mg). Antibacterial assay Compounds 1 and 2 were tested for antibacterial activity using a disk diffusion assay [10]. Seed cultures of three bacteria, Staphylococcus aureus ATCC29213, Bacillus thuringiensis ATCC39765, and Bacillus subtilis ATCC6633, were cultivated in LB broth medium by incubating the organisms for 10 h at 37 °C. Then the seed cultures were added to LB agar medium to make plates. Sterile filter disks (6 mm diameter) infused with 5 mL of test solution ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
S71
(2 mg mL1 DMSO) and positive control (5 mg mL1 DMSO ampicillin) were added to the plates. The plates were placed in an incubator at 37 °C. After 12 h, the diameters of the zones of growth inhibition around each disk were recorded. (1) white powder; m.p. 159–158 °C; ½a25 D 24:1 (c 0.07, MeOH), UV (CH3OH) lmax (log e): 214 (4.50) nm; IR (KBr) nmax: 3447, 3289, 2926, 1740, 1685, 1643, 1537, 975 cm1; 1 H and 13C NMR spectroscopic data (see Table 1); (þ)-HRESIMS m/z 356.1835 [MþNa]þ (calcd. for C18H27NO6Na, 356.1838). (2) Yellow powder; m.p. 131–132 °C; ½a25 D 36:3 (c 0.08, MeOH), UV (CH3OH) lmax (log e): 215(4.12) nm; IR (KBr) nmax: 3391, 3302, 2926, 1689, 1669, 1630, 1551, 1036 cm1; 1H and 13C NMR spectroscopic data (see Table 1); (þ)-HRESIMS m/z 402.1889 [MþNa]þ (calcd. for C20H29NO6Na, 402.1893).
Results and discussion Compound 1 was isolated as a colorless powder. The molecular formula was determined by HRESIMS as C18H27NO6, which suggested 6° of unsaturation. The 1 H, 13C, and DEPT NMR spectra indicated the presence of two methyl (oxygenated), five methylenes, eight methines (two olefinic and four oxygenated), and three quaternary carbons (a ketone carbonyl, an amide carbonyl, and one oxygenated). Accordingly, a bicyclic structure was required for 1 to fulfill the unsaturation requirement. The 1H–1H COSY spectrum (Fig. 2) revealed four 1H–1H spin systems of H-5/H-6/H-7/H-8, H-1/H-2/H-3, H-2/NH, and H-1/OH. The aforementioned data, along with key HMBC correlations (Fig. 2) originated from H-1, H2-3, OH-4, H-5, H-7, and H-8, led to the determination of the bridged cyclic structure. The location of the methoxy group was indicated by the HMBC correlations from OCH3 to C-7, and H-7 to 7-OCH3. The carboxyamide side chain was inferred upon detailed 1H–1H COSY and HMBC spectroscopic analysis. The HMBC correlation of H-2 to C-10 located the carboxyamide side chain at C-2. Taking the molecular formula and the degrees of unsaturation as well as the observation of corresponding resonances for CH-5 and CH-6, the presence of a cis-epoxide was suggested in the structure. By combining all this evidence and data, we were able to assign the gross structure of 1. The geometry of C-20 /C-30 double bond was determined to be E, on the basis of NOESY correlations of H-20 to H-40 . The relative configuration of 1 was deduced from NOESY spectroscopic data and 1H NMR coupling constants. The NOESY cross-peak between H-5 and H-6 and a coupling constant of 3.7 Hz indicated the cis-epoxide ring. The
www.jbm-journal.com
J. Basic Microbiol. 2014, 54, S70–S73
S72
Dong-Ze Liu et al.
Table 1. 1H (500 MHz) and
13
C (125 MHz) NMR Data for 1 and 2 (acetone-d6). 1
No.
dH
1 2 3
4.38 4.74 2.30 2.01
(m) (m) (ddd,12.6,5.0,1.7) (t,12.6)
3.53 3.45 3.87 3.06
(dd,3.7,1.2) (dd,3.7,1.5) (dd,7.5,1.2) (ddd,7.5,3.4,1.5)
6.04 6.74 2.15 1.44 1.29 1.28 0.86
(dt,15.1, 1.5) (dt,15.1, 7.0) (m) (m) (m) (m) (t, 7.0)
4.62 4.48 3.48 7.97
(d, 3.6) (s) (s) (br d, 8.1)
4 5 6 7 8 9 10 20 30 4 50 60 70 80 90 100 OH-1 OH-4 OCH3 NH
2 dC 71.3 46.3 38.1 75.7 63.8 55.4 77.5 55.1 207.3 164.8 124.2 143.8 31.6 28.3 31.8 22.4 13.6
57.5
large coupling constant (12.6 Hz) suggested a trans-diaxial orientation of H-2 and H-3b. A W-type long-range coupling (J ¼ 1.7 Hz) was observed between two equatorial protons, H-1 and H-3a. Irradiation of H-7 in a NOEDIFF experiment resulted in signal enhancement of H-1, while irradiation of H-3a gave signal enhancement of H-5. From the above analysis, the structure of 1 was established as shown.
Figure 2. Key COSY and HMBC correlations in 1 and 2. ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
dH
dC
5.20 4.52 1.98 1.81
(dd,4.4, 3.8) (m) (dd,13.5,4.5) (t,13.5)
4.09 4.26 6.90 5.89
(d,8.3) (dt,8.3,2.1) (dd,10.5,2.1) (dd, 10.5, 2.1)
6.05 6.74 2.16 1.55 1.32 1.98 5.43 5.43 1.60 5.81 4.93 3.47 6.88
(dt,15.1, 1.5) (dt,15.1, 7.0) (m) (m) (m) (m) (m) (m) (m) (br d, 4.4) (s) (s) (br d, 8.9)
90.2 44.8 30.1 72.7 73.5 76.3 148.4 126.3 196.2 164.7 124.3 143.6 31.5 27.7 28.7 32.3 131.8 124.1 17.2 57.7
Compound 2 was obtained as yellow amorphous powder. Its molecular formula was established as C20H29NO6 by HRESIMS data (m/z 402.1889 [MþNa]þ, calcd. 402.1893), which suggested 7° of unsaturation. The IR spectrum showed absorption bands at 3380 (OH), 1689 (conjugated ketone), and 1669 (amide carbonyl) cm1. The 1H, 13C, and DEPT NMR spectra indicated the presence of 2 methyl (oxygenated), 5 methylenes, 10 methines (6 olefinic, a hemiacetal, and 2 oxygenated), and 3 quaternary carbons (a ketone carbonyl, an amide carbonyl, and 1 oxygenated). Accordingly, a bicyclic structure was required for 2 to fulfill the unsaturation requirement. The 1H–1H COSY spectrum (Fig. 2) revealed four 1H–1H spin systems of H-5/H-6/H-7/H-8, H-1/H-2/H-3, H-2/NH, and H-1/OH. The aforementioned data, along with key HMBC correlations (Fig. 2) originated from H-1, H2-3, OH-4, H-5, H-7, and H-9, led to the determination of the bicyclic core. The location of the methoxy group was indicated by the HMBC correlations from OCH3 to C-6, and H-6 to 6-OCH3. The carboxyamide side chain was inferred upon detailed 1H–1H COSY and HMBC spectroscopic analysis. The HMBC correlation of H-2 to C-10 located the carboxyamide side chain at C-2. By combining all this evidence and data, we were able to assign the gross structure of 2. The geometries of the C-7/C-8 and C20 /C-30 double bonds were determined to be Z and E, respectively, on the basis of NOESY correlations of H-7 to
www.jbm-journal.com
J. Basic Microbiol. 2014, 54, S70–S73
New alkaloids from Ramaria madagascariensis
H-8 and H-20 to H-40 . Due to the overlap of the olefinic proton resonances, trans configuration (80 E) was addressed by comparison of the chemical shifts of C-70 C-100 with corresponding carbons for trans-2-heptene (C1 dC 17.9, C-2 dC 124.6, C-3 dC 131.7, C-4 dC 32.4) and cis-2heptene (C-1 dC 12.7, C-2 dC 123.7, C-3 dC 130.9, C-4 dC 26.7). The relative configuration of 2 was deduced on the basis of 1H–1H J values and NOESY spectroscopic data. A NOESY cross-peak observed between H-3b and H-5 and the large coupling constant, J2,3b of 13.5 Hz, indicated the axial orientations of H-2, H-3b, and H-5, and trans ring junction, while the 1H–1H J value, J1,2 of 3.8 Hz, suggested an equatorial position of H-1. The large coupling constant for H-5 and H-6 (J ¼ 8.3 Hz) suggested b-position of H-5 and a-position of H-6. From the above evidence, the structure of 2 was established as shown. Structurally, compounds 1 and 2 possess chemical skeletons similar to isariotins E-J and TK-57-164A [10–14] except for the carboxyamide side chain or chloro substituent on the cyclic core. Accordingly, biosynthetic routes to compounds 1 and 2 may resemble those for the above compounds starting with condensation of the corresponding unsaturated carboxylic acid and L-tyrosine [14]. Compounds 1 and 2 were evaluated for their antimicrobial activity. However, none of them showed an inhibition zone at a concentration of 100 mg/disk.
References [1] Barros, L., Calhelha, R.C., Vaz, J.A., Ferreira, I.C.F. et al., 2007. Antimicrobial activity and bioactive compounds of Portuguese wild edible mushrooms. Eur. Food. Res. Technol., 225, 151–156. [2] Barro, L., Baptista, P., Estevinho, L.M., Ferreira, I.C.F., 2007. Bioactive properties of the medicinal mushroom Leucopaxillus giganteus mycelium obtained in the presence of different nitrogen sources. Food Chem., 105, 179–186. [3] Li, S.P., Zhang, G.H., Zeng, Q., Huang, Z.G. et al., 2006. Hypoglycemic activity of polysaccharide, with antioxida-
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
S73
tion, isolated from cultured Cordyceps mycelia. Phytomedicine, 13, 428–433. [4] Xiao, J.H., Xiao, D.M., Chen, D.X., Xiao, Y. et al., 2012. Polysaccharides from the medicinal mushroom Cordyceps taii show antioxidant and immunoenhancing activities in a D-galactose-induced aging mouse model. Evid.-Based Complement. Altern. Med., 2012, 273–435. [5] Xu, T., Beelman, R.B., Lambert, J.D., 2012. The cancer preventive effects of edible mushrooms. Anticancer Agents Med. Chem., 12, 1255–1263. [6] Liu, D.Z., Dong, Z.J., Wang, F., Liu, J.K., 2010. Two novel norsesquiterpene peroxides from basidiomycete Steccherinum ochraceum. Tetrahedron Lett., 51, 3152–3153. [7] Liu, D.Z., Wang, F., Liao, T.G., Tang, J.G. et al., 2006. Vibralactone: a lipase inhibitor with an unusual fused bLactone produced by cultures of the basidiomycete Boreostereum vibrans. Org. Lett., 25, 5749–5752. [8] Liu, D.Z., Wang, F., Liu, J.K., 2007. Sesquiterpenes from cultures of the basidiomycete Conocybe siliginea. J. Nat. Prod., 70, 1503–1506. [9] Liu, D.Z., Jia, R.R., Wang, F., Liu, J.K., 2008. A new spiroaxane sesquiterpene from cultures of the basidiomycete Pholiota adiposa. Z. Naturforsch., 63b, 111–113. [10] Wang, X., Filho, J.G.S., Hoover, A.R., King, J.B. et al., 2010. Chemical epigenetics alters the secondary metabolite composition of guttate excreted by an Atlantic-forestsoil-derived Penicillium citreonigrum. J. Nat. Prod. 73, 942–948. [11] Asai, T., Chung, Y.M., Sakurai, H., Ozeki, T. et al., 2012. Highly oxidized ergosterols and isariotin analogs from an entomopathogenic fungus, Gibellula formosana, cultivated in the presence of epigenetic modifying agents. Tetrahedron, 68, 5817–5823. [12] Bunyapaiboonsri, T., Yoiprommarat, S., Srisanoh, U., Choowong, W. et al., 2011. Isariotins G–J from cultures of the Lepidoptera pathogenic fungus Isaria tenuipes. Phytochem. Lett., 4, 283–286. [13] Cha, J.Y., Huang, Y.D., Pettus, T.R.R., 2009. Total synthesis of TK-57-164A, isariotin F, and their putative progenitor isariotin E. Angew. Chem. Int. Ed., 48, 9519–9521. [14] Bunyapaiboonsri, T., Yoiprommarat, S., Intereya, K., Rachtawee, P. et al., 2009. Isariotins E and F, spirocyclic and bicyclic hemiacetals from the entomopathogenic fungus Isaria tenuipes BCC 12625. J. Nat. Prod., 72, 756–759.
www.jbm-journal.com
J. Basic Microbiol. 2014, 54, S70–S73
