Discovery of tryptamine derivatives from Bacillus sp. PKU-TA00001
Tongtong Geng, Guiyang Wang, Xueyang Ma, Zhongyi Zhang, Tan Liu, Yuanjie Ge, Jing Jin, Xiaoxu Sun, Yingtao Zhang, Donghui Yang*, Ming Ma*
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
Abstract: Tryptamine-derived natural products have been discovered from different sources including animals, plants and bacteria,and they show various biological activities. However, they are not discovered widely compared with the large amounts of tryptaminederivatives generated by chemical synthesis. We here report the discovery of five tryptamine-derived natural products (1–5) and one known polyketide 6 from Bacillus sp. PKU-TA00001. Compounds 1 and 2are new compounds featuring methyl-hexanamide moieties, compound 4is first discovered as a natural product, and 3 and 4’s NMR data are first provided. All compounds showed MIC (minimum inhibitory concentration) values higher than 50 µM against several Gram-positive and negative strains, and showed no cytotoxicity at the concentration of 100 µM against the human cancer cell lines A549, HCT-8 and MCF-7. The discovery of 1–4expands the structural diversity of tryptamine-derived natural products, and sets the stage for revealing their biosynthetic mechanisms and biological activities in the future.
Keywords: Bacillus;Strain prioritization; Tryptamine-derived natural products
CLC number: R284 Document code: A Article ID: 1003–1057(2019)8–527–10
Tryptamine is an important natural product featuring an indole ring, and it is biosynthesized from the decarboxylation of tryptophan. It is the early intermediate in the biosynthesis of monoterpenoid indole alkaloids, such as vinblastine, reserpine and strychnine, and its naturally occurring-derivatives in animals, plants and bacteria stand as important bioactive molecules. Serotonin (5-hydroxytryptamine) is one of the most important signaling hormones in animal body and involved in multiple processes in the central nervous system. Melatonin (N-acetyl-5-methoxytryptamine) is a pineal gland-released hormone involved in the regulation of normal circadian rhythm in mammals and also possesses anti-oxidation activity. Despite of the simple structures of serotonin and melatonin, many of their derivatives have been synthesized to be used as potential psychoactive molecules[2,5]. Tryptamine-derived natural products are also discovered from marine animals, such as granulatamides A and B featuring fatty acid amide moieties from the soft coral Eunicella granulata, and leptoclinidamide and (–)-leptoclinidamine B featuring amino acid-substitutionsfrom the Indonesian ascidian Leptoclinides dubius. In plants, Rollinia mucosa and Annona atemoya produce a series of N-fatty acyl tryptamines in their seeds[8,9]. In bacteria, Bacillus and Xenorhabdus strains are importantproducers of tryptamine-derived natural products, exemplified by bacillamides A–C featuring a thiazole ring from Bacillus endophyticus[10–12], bacilsubteramide Afeaturing a hydroxy group attached to C-3 from Bacillussubterraneus 11593, and nematophin featuring an α-keto-acyl moiety from Xenorhabdus nematophilus.
Although tryptamine-derived natural products can be produced from different sources including animals, plants and bacteria, they are not discovered widely compared with the large amounts of tryptamine derivatives generated by chemical synthesis[2,15–18]. We here report the discovery of five tryptamine-derived naturalproducts 1–5 and one known polyketide 6 fromBacillus sp. PKU-TA00001. Compounds 1 and 2are new compounds, and their structures are elucidated based on 1D and 2D NMR, HRESIMS and Mosher ester analyses. Compared with known tryptamines containing fatty acid amide moieties, compounds 1 and 2contain different methyl-hexanamide moieties, expanding the structural diversity of tryptamine-derived natural products. Compound 4is first discovered as a natural product, compound 3is first discovered from Bacillus, and 3 and 4’s NMR data are first provided. These results set the stage for revealing their biosynthetic mechanisms and biological activities in the future.
2.1. General experimental procedures
Optical rotations were determined on an Autopol III automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). 1H and 13C NMR spectra were recorded on an Avance III 400 NMR spectrometer (Bruker Corporation, Billerica, USA). HRESIMS spectra were obtained from an IT-TOF LC/MS spectrometer (Shimadzu, Kyoto, Japan). HPLC analysis was performedon an Agilent 1260 series (Agilent Technologies, Santa Clara, USA) with a YMC-Pack ODS-A column (250 mm×4.6 mm, 5 μm, YMC Co., Ltd., Shimogyo-ku, Kyoto, Japan). Semi-preparative HPLC was performed on an SSI 23201 system (Scientific Systems Inc., StateCollege,USA) with a YMC-Pack ODS-A column (250 mm×10 mm, 5 μm, YMC Co., Ltd., Shimogyo-ku, Kyoto, Japan). MPLC was performed on an LC3000 series(Beijing Tong Heng Innovation Technology, Beijing, China) with a ClaricepTM Flash i-series C18 cartridge (20–35 μm, 40 g, Bonna-Agela,Wilmington, DE, USA). The fermentations were carried out with MQD-B1R shakers (Minquan Instrument Co., Ltd., Shanghai, China).
2.2. Strains isolation
The strain PKU-TA00001 was isolated from a soil sample collected from the grassplot in Boshan county (36°34? N, 117°96? E), Zibo city, Shandong province, China. The isolation of the strain was carried outaccording to the published procedures. Briefly, about 1 cm3 of the soil sample was homogenized in 9 mL sterilized PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 in 1.0 L distilled water, pH 7.4). The homogenates were incubated in shaker at 28 °C, 220 r/min for 30 min, pretreated by incubating in a water bath at 55 °C for 6 min, and put into biosafety cabinet for 30 min. The supernatants (200 µL for each plate) were plated in triplicate on agar plates. Four different media, TI-1 (soluble starch 20 g, KNO3 1 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, FeSO4·7H2O 0.01 g, agar 15 g in 1.0 L distilled H2O, pH 7.2–7.4), TI-2 (soluble starch 20 g, CaCO3 2 g, (NH4)2SO4 2 g, K2HPO4 1 g, MgSO4·7H2O 1 g, NaCl 1 g,FeSO4·7H2O 0.001 g, MnCl2·7H2O 0.001g, ZnSO4·7H2O 0.001 g, agar 15 g in 1.0 L distilled H2O, pH 7.2), TI-3 (asparagine 1 g, glycerol 10 g, K2HPO4 1 g, FeSO4 0.001 g, MnCl2 0.001 g, ZnSO4 0.001 g, agar 15 g in 1.0 L distilled H2O, pH 7.2), and TI-4 (p-aminobenzoic acid 0.5 mg, inositol, 0.5 mg, nicotinic acid 0.5 mg, riboflavin 0.5 mg, pantothenic acid 0.5 mg, vitamin B1 0.5 mg, vitamin B6 0.5 mg, biotin 0.25 mg, humic acid 1 g, Na2HPO4 0.5 g, KCl 1.7 g, MgSO4·7H2O 0.05 g, FeSO4·7H2O 0.01 g, CaCl2 1 g, agar 15 g in 1.0 L distilled H2O, pH 7.2), were used for strain isolation. Each agar plate was supplemented with cycloheximide (50 µg/mL) and nalidixic acid (50 µg/mL) to suppress the growth fungi and fast-growing Gram-negative bacteria, respectively. The single colonies of strain PKU-TA00001were picked from the TI-2 agar plate after incubation at 28 °C for 20 d, and purified after being re-streaked on agar plates for several times. Using above-method procedures, 412 bacterial strains were isolated from 20 soil samples collected in different areas in Boshan county, China.
2.3. Small-scale fermentation and HPLC analysis
Spore suspension (50 µL) of Bacillus sp. PKU-TA00001 was inoculated into 50 mL of seed medium MI-4 (yeast extract 1 g, peptone 5 g, beef extract 1 g, FePO40.01 g, in 1.0 L distilled H2O, pH 7.4), and incubated in a shaker at 28 °C, 220 r/min for 3 d. Two milliliters of the resultant seed culture was inoculated into 50 mL of production medium TP-1 (soluble starch 20 g, glucose 10 g, peptone 5 g, yeast extract 5 g, NaCl 4 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, CaCO3 2 g in 1.0 L distilled water, pH 7.0), and the fermentation continued at28 °C, 220 r/min for 7 d. The D101 (3 g/50 mL) resins were added 12 h before the fermentationfinished. The resin and cell mass were harvested by centrifugation, washed with distilled H2O and extracted with MeOH. The MeOH extract of Bacillus sp. PKU-TA00001 was concentrated and analyzed by HPLC. The HPLC analysis was carried out with a flow rate of 1 mL/min with UV detection at 280 nm, using a gradient elution program from 5% MeOH in H2O to 100% MeOH over 28 min.
2.4. Phylogenetic analysis of strain PKU-TA00001
The genomic DNA of Bacillus sp. PKU-TA00001 was extracted by using salting out method. The housekeeping 16S rRNA genes of the strain were amplified by PCR using the primers 27F (5′-AGAGTTTGATCMTGGC-TCAG-3′) and 1492R (5′-TACGGYTACCTTGTTA-CGACTT-3′). The 20-μL PCR system consisted of 10 μL 2×M5 HighGC Taq (Beijing, Mei5 Biotech Beijing, China), 1 μL the forward primer, 1 μL the reverse primer, 2 μL genomic DNA, 3.5 μL ddH2O, and 1.5 μL DMSO. The PCR program was performed with an initial denaturation for 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C/30 s at 52 °C/1 min 30 s at 72 °C, and a final extension for 5 min at 72 °C. The PCR products were analyzed by electrophoresis on a 1% (w⁄v) agarose gel and recovered using a gel purificationkit. The recovered PCR products were sequenced. The phylogenetic tree was generated with Mega 6.0 (Pennsylvania State University, State College, PA, USA) using the Neighbor-Joining algorithm, based on the 16S rRNA gene (GeneBank accession number MK640615.1) of strain PKU-TA00001 and homologous genes searched by BLAST on the NCBI website.
2.5. Large-scale fermentation and isolation of 1–6
The large-scale fermentation (6 L) was carried out in same procedures as the small-scale fermentation. The resins and cell mass were harvested by centrifugation and extracted with MeOH. The MeOH extract was concentrated, resuspended in H2O and extracted with EtOAc for three times. The EtOAc extract (2.4 g) was fractionated by MPLC using a gradient elution from 5% MeOH in H2O to 100% MeOH over 40 min, with the flow rate of 15 mL/min under the UV detection at 210 nm, to give 50 fractions (a1–a50). Fractions a28–a30were combined based on HPLC analysis and subjected to Sephadex LH-20 column chromatography eluted with MeOH to give 30 fractions (b1–b30). Fractions b4–b13 was combined and further purified by semi-preparative HPLC eluted with CH3CN/H2O (35/65, v/v) with the flow rate of 2 mL/min under the UV detection at 210 nm, to afford 1 (1.12 mg) and 4 (2.44 mg). Fractions a34 and a35 were combined based on HPLC analysis and subjected to Sephadex LH-20 column chromatography eluted with MeOH to give 27 fractions (c1–c30). Fractions c11–c15 were combined and further purified by semi-preparative HPLC eluted with CH3CN–H2O (40:60, v/v) with the flow rate of 2 mL/min under the UV detection at 210 nm to afford 2 (2.25 mg) and 3 (1.84 mg); and fractions c21–c23 were combined and further purified by semi-preparative HPLC eluted with CH3CN–H2O (35:65, v/v) with the flow rate of 2 mL/minunder the UV detection at 210 nm to afford 5 (1.39 mg). Fractions a36 was subjected to Sephadex LH-20 columnchromatography eluted with MeOH to give 15 fractions (d1–d15). Fractions d7–d11 were combined and further purified by semi-preparative HPLC eluted with CH3CN–H2O (55:45, v/v) with the flow rate of 2 mL/min under the UV detection at 210 nm to afford 6 (2.35 mg).
2.6. Mosher ester analysis of compounds 1 and 4
Compound 1 or 4 (0.5 mg for each)was dissolved in 200 μL of CH2Cl2, and S-MTPA-Cl (6 equiv., 10 μL) orR-MTPA-Cl (6 equiv., 10 μL), 4-dimethylaminopyridine(DMAP, 0.2 mg, 1 equiv.) and triethylamine (10 equiv.,10 μL) were added to the solution under N2 gas protection. The reaction was stirred overnight (16 h) at room temperature[22,23]. The products were purified by semi-preparative HPLC eluted with a program from 5%CH3CN in H2O to 100% CH3CN over 28 min, with the flow rate of 2 mL/min under the UV detection at 280 nm, and sent to the 1H NMR experiments.
3.1. Strain prioritization based on HPLC analysis and phylogenetic analysis of strain PKU-TA00001
Strain PKU-TA00001 was one of the 412 strains isolated from 20 soil samples collected from Boshan county, Shandong province, China (Experimental 2.2). We fermented these isolated strains in small scales (50 mL), and analyzed their crude extracts by HPLC. The crude extract of PKU-TA00001 fermented in medium TP-1 showed abundant natural products under the UV detection at 280 nm. Therefore, we selected PKU-TA00001 as a prioritized strain for the large-scalefermentation to isolate natural products. PKU-TA00001was classified as a Bacillus species on the basis ofa phylogenetic analysis by the comparison of its housekeeping 16S rRNA gene with those from other homologues (Fig. 1).
Figure 1. The phylogenetic analysis of Bacillus sp. PKU-TA00001 (labeled in red). The strain streptomyces hygroscopicus A-229 was used as an outgroup. The GenBank accession numbers were shown in parentheses.
A large-scale fermentation (6 L) of Bacillus sp. PKU-TA00001 was carried out in medium TP-1. Six compounds (1–6) were isolated with a combination of chromatographic methods. Their structures were elucidatedas (S)-3-hydroxy-N-[2-(1H-indol-3-yl) ethyl]-5-methyl-hexanamide (1), N-[2-(1H-indol-3-yl) ethyl]-4-methyl-hexanamide (2), N-[2-(1H-indol-3-yl) ethyl]-5-methyl-hexanamide (3), (R)-3-hydroxy-N-[2-(1H-indol-3-yl) ethyl]-4-methyl-pentanamide (4), 1-acetyl-β-carboline (5) and basiliskamide A (6), based on 1D and 2D NMR, MS and Mosher ester analyses (Fig. 2).
Figure 2. The structures of compounds 1–6 discovered from Bacillus sp. PKU-TA00001.
3.2. Structural elucidation of compounds 1–6
3.2.1. (S)-3-Hydroxy-N-[2-(1H-indol-3-yl) ethyl]-5-methyl-hexanamide (1)
Colorless oil, –8.48 (c 0.10, MeOH); HRESIMS analysis afforded an [M-H]– ion at m/z 287.1759 (calcd for C17H23N2O2, 287.1759), giving the molecular formulaof 1 as C17H24N2O2. UV (MeOH) λmax (log ε) 222 (4.41), 281 (3.64) nm. IR νmax 3397, 2959, 2931, 1680, 1634, 1204, 1139, 744 cm–1. The 1H NMR and 13C NMR spectra of 1 resembled those of Nb-acetyltryptamine except that the resonances at δH 1.92 and δC 23.4 attributed to the methyl group in Nb-acetyltryptamine were replaced by resonances at δH 2.13 (d, J = 7.0 Hz, H2-12), 3.87 (br s, H2-13), 4.60 (d, J = 5.3 Hz, 13-OH), 1.26 (m, Ha-14), 1.09 (m, Hb-14), 1.72 (m, H2-15), 0.86 (d, J = 6.3 Hz, H3-16), 0.84 (d, J = 6.3 Hz, H3-17) in the 1H NMR spectrum of 1, and resonances at δC 44.4 (C-12), 65.5 (C-13), 46.2 (C-14), 23.9 (C-15), 23.5 (C-16) and 21.8 (C-17) in the 13C NMR spectrum of 1, respectively. These comparisons suggested that 1 contained the same acylated tryptamine moiety as that in Nb-acetyltryptamine, but contained a different acyl group attached to the nitrogen. The new occurring proton signals in the1H NMR spectrum of 1, compared to those in the 1H NMRspectrum of Nb-acetyltryptamine, constituted a coupling system from -CH2-CH(OH)-CH2-CH(CH3)2, based on the COSY spectrum of 1. This suggested that 1 contained a 3-hydroxy-5-methyl-hexanamide moiety, which was confirmed by the HSQC and HMBC spectra of 1. The acylation at the N-10 was established by the correlations from H2-9 and H-10 to C-11 in the HMBC spectrum. Therefore, the planar structure of 1was identified as 3-hydroxy-N-[2-(1H-indol-3-yl) ethyl]-5-methyl-hexanamide.
To identify the absolute configuration of C-13, the Mosher ester analysis was carried out. The S- and R-MTPA (α-methoxy-α-trifluoromethylphenylacetic acid) esters of 1 were prepared[22,23], respectively, and their 1H NMR spectra were recorded. The positive ΔδSR (ΔS-ΔR) values for H2-12, N-10, H2-9, H2-8 and H-2, in combination with the negative ΔδSR values for H2-14, H-15, H3-16 and H3-17, establishing the absolute configuration of C-13 as S. Therefore, the structure of 1 was identified as (S)-3-hydroxy-N-[2-(1H-indol-3-yl) ethyl]-5-methyl-hexanamide (Fig. 3).
3.2.2. N-[2-(1H-Indol-3-yl) ethyl]-4-methyl-hexanamide (2)
Colorless oil, +40.0 (c 0.10, MeOH), HRESIMS analysis afforded an [M-H]– ion at m/z 271.1811 (calcd for C17H23N2O, 271.1810), giving the molecular formulaof 2 as C17H24N2O. UV (MeOH) λmax (log ε) 221 (4.56), 280 (3.86) nm. IR νmax 3398, 2921, 1682, 1207, 1139, 1024, 801, 722 cm–1. Analysis of the 1H and 13C NMR spectra of 2 showed that it contained a same acylated tryptamine moiety as that in 1, but contained a differentacyl group attached to N-10. The COSY spectrum of 2showed the proton signals at δH 2.04 (t, J = 7.3 Hz, H2-12), 1.29 (m, H2-13), 1.53 (m, H2-14), 1.11 (m, H-15), 0.83 (t, J = 7.0 Hz, H3-16) and 0.83 (d, J = 5.0Hz, H3-17), constituting a coupling system from -CH2-CH2-CH(CH3)-CH2-CH3. This suggested that 2 contained a 4-methyl-hexanamide moiety, which was confirmed by the HSQC and HMBC spectra of 2. The acylation at the N-10 was established by the correlations from H2-9 and H-10 to C-11 in the HMBC spectrum. The absolute configuration of C-15 was not identified. Therefore, the structure of 2 was identified as N-[2-(1H-indol-3-yl) ethyl]-4-methyl-hexanamide.
3.2.3. N-[2-(1H-Indol-3-yl) ethyl]-5-methyl-hexanamide (3)
Colorless oil, HRESIMS analysis afforded an [M-H]– ion at m/z 271.1811 (calcd for C17H23N2O, 271.1810), giving the molecular formula of 3 as C17H24N2O. Analysisof the 1H and 13C NMR spectra of 3 showed that it contained a same acylated tryptamine moiety as that in 1, but contained a different acyl group attached to N-10. The COSY spectrum of 3 showed the proton signals at δH 2.02 (t, J = 7.4 Hz, H2-12), 1.48 (m, H2-13), 1.11 (m, H2-14), 1.46 (m, H-15), 0.84 (d, J = 6.6 Hz, H3-16 and H3-17), constituting a coupling system from -CH2-CH2-CH2-CH(CH3)2. This suggested that 3 contained a 5-methyl-hexanamide moiety, which was confirmed by the HSQC and HMBC spectra of 3. The acylation at the N-10 was established by the correlations from H2-9 and H-10 to C-11 in the HMBC spectrum. Therefore, the structure of 3 was identified as N-[2-(1H-indol-3-yl) ethyl]-5-methyl-hexanamide. Compound 3 was identified in Xenorhabdusdoucetiae from feeding experiments only based on MS analysis, and we here first provided its NMR data (Table 1 and 2).
Table 1. The 1H NMR (400 MHz) data (J in Hz) of compounds 1–4 in DMSO-d6.
* These signals overlapped with others.
Table 2. The 13C NMR (100 MHz) data (δC, type) of compounds 1–4 in DMSO-d6.
3.2.4. (R)-3-Hydroxy-N-[2-(1H-indol-3-yl) ethyl]-4-methyl-pentanamide (4)
Colorless oil, –10.9 (c 0.10, MeOH). HRESIMS analysis afforded an [M-H]– ion at m/z 273.1602 (calcd for C16H21N2O2, 273.1603), giving the molecular formula of 4 as C16H22N2O2. Analysis of the 1H and 13C NMR spectra of 4 showed that it contained a same acylated tryptamine moiety as that in 1, but contained a differentacyl group attached to N-10. The COSY spectrum of4 showed the proton signals at δH 2.13 (t, J = 8.0 Hz, H2-12), 3.63 (br s, H-13), 4.60 (br s, 13-OH), 1.52 (m, H-14), 0.83 (d, J = 6.8 Hz, H3-15) and 0.81 (d, J = 6.8 Hz, H3-16), constituting a coupling system from -CH2-CH(OH)-CH(CH3)2. This suggested that 4 contained a 3-hydroxy-4-methyl-pentanamide moiety, which was confirmed by the HSQC and HMBC spectra of 4. The acylation at the N-10 was established by the correlations from H2-9 and H-10 to C-11 in the HMBC spectrum.
To identify the absolute configuration of C-13, the Mosher ester analysis was carried out. The S- and R-MTPA (α-methoxy-α-trifluoromethylphenylacetic acid) esters of 4 were prepared, respectively, and their 1H NMR spectra were recorded. The positive ΔδSR values for H2-12,N-10, H2-9, H2-8 and H-2, in combination with the negative ΔδSR values for H-14, H3-15 and H3-16, establishing the absolute configuration of C-13 as R. Therefore, the structure of 4 was identified as (R)-3-hydroxy-N-[2-(1H-indol-3-yl) ethyl]-4-methyl-pentanamide. Compound4 is available as a commercial substance, but there is no any preparation and spectroscopic informations reported.We here first isolated 4 as a natural product and provide its NMR data.
3.2.5. 1-Acetyl-β-carboline (5)
White powder, ESIMS analysis afforded an [M-H]–ion at m/z 209.07. 1H NMR (400 MHz, DMSO-d6) δH: 11.90 (s, NH), 8.52 (d, J = 4.9 Hz, H-3), 8.45 (d,J = 5.0 Hz, H-4), 8.31 (d, J = 7.8 Hz, H-5), 7.82 (br d, J = 8.2 Hz, H-8), 7.60 (ddd, J1 = 8.2 Hz, J2 = 7.2 Hz, J3 = 1.2 Hz, H-6), 7.31 (ddd, J1 = 7.8 Hz, J2 = 7.2 Hz, J3 = 1.0 Hz, H-7), 2.80 (s, H3-2′). 13C NMR (100 MHz, DMSO-d6) δC: 142.3 (C-1), 137.9 (C-3), 119.9 (C-4), 120.6 (C-4a), 131.4 (C-4b), 122.3 (C-5), 129.3 (C-6), 120.30 (C-7), 113.3 (C-8), 136.4 (C-8a), 134.4 (C-9a), 201.5 (C-1′), 26.38 (C-2′). These data were consistent with those of authentic 1-acetyl-β-carboline.
3.2.6. Basiliskamide A (6)
Colorless oil, –80.0 (c 0.10, MeOH). HRESIMS analysis afforded an [M+Na]+ ion at m/z 408.2152(calcd for C23H31NO4Na, 408.2151), giving the molecularformula of 6 as C23H31NO4. 1H NMR (400 MHz, DMSO-d6) δH: 7.37 (s, 1a-NH), 6.88 (s, 1b-NH), 5.57 (d, J = 11.4 Hz, H-2), 6.33 (dd, J1 = 11.3 Hz, J2 = 11.4 Hz, H-3), 7.43 (m, H-4) 5.93 (dt, J1 = 15.1 Hz, J2 = 4.7 Hz, H-5), 2.30 (dd, J1 = 13.6Hz, J2 = 7.6 Hz, H-6a), 2.00 (m, H-6b), 3.51 (br s, H-7), 2.04 (m, H-8), 4.94 (dd, J1 = 9.7 Hz, J2 = 2.4 Hz, H-9), 1.66 (m, H-10), 1.25 (m, H-11a), 1.13 (m, H-11b), 0.89 (t, J = 8.0 Hz, H3-12), 0.86 (d,J = 7.9 Hz, H3-13), 0.93 (d, J = 4.6 Hz, H3-14), 6.64 (d, J = 16.0 Hz, H-16), 7.66 (d, J = 15.9 Hz, H-17), 7.74 (m, H-19 and H-23), 7.43 (m, H-20, H-21 and H-22), 4.62 (d, J = 5.1 Hz, 7-OH). 13C NMR (100 MHz, DMSO-d6) δC: 168.0 (C-1), 119.8 (C-2), 141.1 (C-3), 128.7 (C-4), 141.1 (C-5), 35.2 (C-6), 70.3 (C-7), 41.3 (C-8), 77.0 (C-9), 36.0 (C-10), 26.9 (C-11), 10.6 (C-12), 12.1 (C-13), 13.3 (C-14), 166.6 (C-15), 118.5 (C-16), 145.1 (C-17), 134.6 (C-18), 128.9 (C-19/23), 129.4 (C-20/22), 130.9 (C-21). These data were consistent with those of authentic basiliskamide A.
Compounds 1–6 were tested for their antibacterial and cytotoxicity activities. The Gram-positive strains Staphylococcus aureus ATCC 29213, Bacillus subtilisATCC 6051, vancomycin-resistant Enterococcus faecalis A4, and Gram-negative strains Escherichia coli ATCC 25922, Pseudomonas aeruginosa 14 and Klebsiella pneumoniae WNX-1, were selected for the antibacterial assays. Compounds 1–6 showed MIC (minimum inhibitory concentration) values higher than 50 µM against all the tested strains. The human cancer cell lines A549, HCT-8 and MCF-7 were selected for the cytotoxicity assays, and 1–6 showed no cytotoxicity at the concentration of 100 µM. More types of assays are needed to reveal potential biological activities of 1–6.
This research was supported in part by the National Natural Science Foundation of China (Grant No. 81673332, 81573326, 81741148).
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Bacillus sp. PKU-TA00001中发现的色胺类天然产物
耿彤彤, 王贵阳, 马学洋, 张中义, 刘谈, 葛元洁, 金晶, 孙晓旭, 张英涛, 杨东辉*, 马明*
摘要: 色胺类天然产物已经在动物、植物和微生物中得到分离并且具有多种生物活性。然而, 相对于大量化学合成的色胺类化合物, 只有有限数量的色胺类天然产物被发现。我们从菌株Bacillus sp. PKU-TA00001中发现了五个色胺类天然产物(1–5), 其中化合物1和2是新化合物, 化合物4是新天然产物, 化合物3和4的核磁共振波谱数据首次被提供。生物活性筛选结果显示, 所有化合物对几株革兰氏阳性菌和阴性菌的最低抑制浓度(MIC)均在50 µM以上, 对人肿瘤细胞株A549, HCT-8和MCF-7在100 µM下未显示细胞毒活性。化合物1–4的发现扩展了色胺类天然产物的结构多样性, 并为将来的生物合成和更多生物活性研究打下了基础。
关键词: Bacillus; 菌株优选; 色胺类天然产物
Received: 2019-04-19; Revised: 2019-05-12; Accepted: 2019-05-26.
Foundation items: National Natural Science Foundation of China (Grant No. 81673332, 81573326, 81741148).
*Corresponding author. Tel.: +86-10-82801559; +86-10-82805794, E-mail: firstname.lastname@example.org