Article pubs.acs.org/JAFC
Non-cytotoxic Antifungal Agents: Isolation and Structures of Gageopeptides A−D from a Bacillus Strain 109GGC020 Fakir Shahidullah Tareq,†,‡ Min Ah Lee,‡ Hyi-Seung Lee,‡ Yeon-Ju Lee,‡ Jong Seok Lee,‡ Choudhury M. Hasan,§ Md. Tofazzal Islam,∥ and Hee Jae Shin*,†,‡ †
Department of Marine Biotechnology, Korea University of Science and Technology, Daejeon 350-360, Republic of Korea Marine Natural Products Laboratory, Korea Institute of Ocean Science and Technology, Ansan 426-744, Republic of Korea § Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh ∥ Department of Biotechnology, Bangabandhu Sheikh Muzibur Rahman Agricultural University, Dhaka 1706, Bangladesh ‡
S Supporting Information *
ABSTRACT: Antifungal resistance and toxicity problems of conventional fungicides highlighted the requirement of search for new safe antifungal agents. To comply with the requirement, we discovered four new non-cytotoxic lipopeptides, gageopeptides A−D, 1−4, from a marine-derived bacterium Bacillus subtilis. The structures and stereochemistry of gageopeptides were determined by NMR data analysis and chemical means. Gageopeptides exhibited significant antifungal activities against pathogenic fungi Rhizoctonia solani, Botrytis cinerea, and Colletotrichum acutatum with minimum inhibitory concentration (MIC) values of 0.02−0.06 μM. In addition, these lipopeptides showed significant motility inhibition and lytic activities against zoospores of the late blight pathogen Phytophthora capsici. These compounds also showed potent antimicrobial activity against Gram positive and Gram negative bacteria with MIC values of 0.04−0.08 μM. However, gageopeptides A−D did not exhibit any cytotoxicity (GI50 > 25 μM) against cancer cell lines in sulforhodamine B (SRB), 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT), and WST-1 ((4-[3−4-iodophenyl]-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene disulfonate)) assays, demonstrating that these compounds could be promising candidates for the development of non-cytotoxic antifungal agents. KEYWORDS: Bacillus subtilis, gageopeptides, antifungal agents, non-cytotoxicity
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INTRODUCTION Fungal diseases are very common crop-devastating diseases in agricultural practice. For example, Rhizoctonia solani (R. solani) is a plant pathogenic fungus with a wide host range and worldwide distribution. This pathogen is very destructive to many crops, including agronomic, ornamental, and forestry species.1 This species has been reported to enormously reduce the growth and quality of crops such as cereals, potato, lettuce, and cotton in Spain.2−5 Phytophthora capsici (P. capsici), another economically important pathogen, causes extensive damage in cucumber, pepper, tomato, and beans. It is well recognized to cause crown, leaf, and fruit blight, wilting of the whole plant, and dark purplish discoloration of the stem.6 Similarly, a diverse group of fungi are recognized to cause severe plant diseases, resulting in a significant loss in agricultural practice.7 Therefore, it is very important to control plant diseases to secure the level of yield both quantitatively and qualitatively. Farmers often heavily rely on the application of commercial fungicides to secure the quality and production of crops. Most of the fungicides are not compatible with the environment or health; their excessive application and misuse can also cause acute and chronic toxicity.8,9 According to a review by the International Labor Organization (ILO), about 14% of all occupational injuries were caused by exposure to synthetic pesticides and other agrochemicals.10 Moreover, the World Health Organization (WHO) and the United Nations Environ© XXXX American Chemical Society
ment Programme (UNEP) reported that three million farmers in the developing countries suffer from severe poisoning by commercial pesticides, resulting in the deaths of about 18,000 of them each year.11 However, the environmental problems caused by synthetic fungicides have brought a significant change to people who work in the agricultural field toward the use of harmful pesticides. Therefore, it has now become an urgent issue to develop environmentally friendly alternative pesticides to synthetic chemicals to protect plants from diseases. Lipopeptides produced nonribosomally by bacteria and fungi have been reported to be biodegradable and less toxic, and have various biomedical applications.12 As illustrations, lipopeptides of surfactins, iturins, mycosubtilin, plipastatin, halobacillin, fengycin, and echinocandin families produced by bacterial origins have recently received much attention due to their potent biological activities, including antimicrobial, antitumor, antiviral, anti-inflammatory, and immunosuppressive activities.13−18 In particular, echinocandins and pneumocandins are now approved antifungal drugs which inhibit the synthesis of glucan in the cell wall, via noncompetitive inhibition of the enzyme 1,3-β-D-glucan synthase in pathogenic fungi including Received: February 13, 2014 Revised: May 25, 2014 Accepted: May 26, 2014
A
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Journal of Agricultural and Food Chemistry
Article
Aspergillus and Candida.19 In our previous studies,20,21 we have discovered three novel lipotetrapeptides, gageotetrins A−C and surfactin like heptapeptides, gageostatins A−C with significant antimicrobial properties. Further investigations on the ethyl acetate (EtOAc) extract obtained from the same bacterium Bacillus subtilis (B. subtillis) afforded four new lipopeptides, gageopeptides A−D, 1−4 (Figure 1), which displayed
sequence of the strain 109GGC020 was submitted to GenBank under accession number JQ927413. A BLAST search result showed the sequence of 109GGC020 to be 100% similar to B. subtilis. Isolation of Compounds 1−4. Batch fermentation (60 L) of the strain B. subtilis 109GGC020 was carried out in 2 L flasks. For 1 L, the medium composition (w/v) was 1% dextrose, 0.1% yeast extract, 0.1% beef extract, 0.2% tryptone, 100% natural seawater, and pH 7.1. The culture broth was separated from the cells by centrifugation after 7 days of cultivation at 28 °C. The broth was extracted with EtOAc and the solvent was removed under reduced pressure at 40 °C to dryness. The EtOAc extract (11.8 g) was fractionated by an ODS open column chromatography using methanol:water (1:4, 2:3, 3:2, 4:1, and 100:0; v/v) as eluent. The 100% MeOH fraction was refractionated and purified by ODS semipreparative and analytical HPLCs with a solvent system of 95% MeOH in H2O to yield 1−4. Gageopeptide A (1; 4.6 mg; 0.04%): amorphous solid; [α]27D −21 (c 0.1, MeOH). IR (MeOH; νmax, cm−1): 3285 (NH), 1620−1689 (CO), 2923 (aliphatic chain). 1H and 13C NMR data (CD3OD): Table 1. HRESIMS (m/z): 735.4883 [M + Na]+. Calcd for C37H68N4O9: 735.4884 [M + Na]+; Gageopeptide B (2; 1.8 mg; 0.02%): amorphous solid; [α]27D −40 (c 0.1, MeOH). IR (MeOH; νmax, cm−1): 3278 (NH), 1642 (CO), 2927 (aliphatic chain). 1H and 13C NMR data (CD3OD): Table 1. HRESIMS (m/z): 763.5192 [M + Na]+. Calcd for C39H72N4O9: 763.5197 [M + Na]+. Gageopeptide C (3; 5.9 mg; 0.05%): amorphous solid; [α]27D −20 (c 0.1, MeOH). IR (MeOH; νmax, cm−1) 3373 (NH), 1625 (CO), 2970 (aliphatic chain). 1H and 13C NMR data (CD3OD): Table 1. HRESIMS (m/z): 735.4883 [M + Na]+. Calcd. for C37H68N4O9: 735.4884 [M + Na]+. Gageopeptide D (4; 7.8 mg; 0.07%): amorphous solid; [α]27D −70 (c 0.05, MeOH). IR (MeOH; νmax, cm−1): 3287 (NH), 1629 (CO), 2959 (aliphatic chain). 1H and 13C NMR data (CD3OD): Table 1. HRESIMS (m/z): 749.5044 [M + Na]+. Calcd for C38H70N4O9: 749.5040 [M + Na]+. Determination of Absolute Stereochemistry of 1−4. The absolute stereochemistry of amino acid residues and at the stereocenter C-3 of 3-hydroxy fatty acids in 1−4 was determined by using the methods described previously.20,21 In brief, compound 1 (1.9 mg) was hydrolyzed using 6 N HCl (500 μL) at 120 °C for 23 h. The hydrolysate was diluted with water and extracted with hexane. The hexane layer was evaporated by a stream of nitrogen, and the water layer was concentrated to dryness under reduced pressure. To determine the absolute configuration at the stereocenter C-3 of the fatty acid,22 the hexane extract was derivatized with Mosher’s reagent and the aqueous extract was used for the determination of absolute stereochemistry of amino acids by Marfey’s method.23 In an entirely analogous way, compounds 2−4 were hydrolyzed and their absolute stereochemistries were assigned. Fatty acid in 1: 1.5 mg; colorless oil. 1H NMR data (CD3OD): δH 2.34 (H-2a, dd, J = 15.0, 8.5 Hz), 2.43 (H-2b, dd, J = 15.5, 5.0 Hz), 3.96 (H-3, m), 1.33 (H-4a, m), 1.45 (H-4b, m), 1.47 (H2-5, m), 1.29 (H2-6−9, brs), 1.18 (H-10, m), 1.50 (H-11, m, overlapped), 1.29 (H12, m, overlapped), 0.86 (H3-13, t, J = 5.5 Hz), 0.87 (H3-14, d, J = 6.5 Hz). LC-MS (m/z): 243.01 [M − H]−. Optical rotation ([α]27D): −26 (c o.1, MeOH). Activity Study of 1−4 against P. capsici. P. capsici was received from Prof. W. Yuanchao of Nanjing Agricultural University, China. Mycelia of P. capsici were cultured on sterile clarified 10% V8 agar in Petri dishes and incubated at 25 °C in the dark for 4 days.24 The agar culture from the Petri dishes incubated for 1−2 weeks was cut into six pieces, covered with sterile distilled water, and cultured in Petri dishes again at 25 °C in the dark to induce sporangium formation. Every 12 h, the water was replaced by fresh sterile distilled water. In this way, after changing the water 3 times, an abundance of sporangia were induced. The Petri dishes with large numbers of sporangia were incubated at 4 °C for 30 min, and kept at room temperature for another 30 min to induce zoospore release. The movement of sporangia and zoospores was checked on a regular basis under an Olympus light microscope. The zoospores remained motile for 10−12
Figure 1. Structures of compounds 1−4.
significant activity against pathogenic fungi and bacteria. The cytoxicity of gageopeptides against human cancer cell lines was also investigated during the study, and the results demonstrated that these new lipopeptides are potent non-cytotoxic antifungal agents.
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MATERIALS AND METHODS
General Experimental Procedures. One- (1D) and twodimensional (2D) NMR data were recorded on a Varian Unity 500 NMR spectrometer (Varian Inc., Palo Alto, CA, USA). IR spectra were measured on a JASCO FT/IR-4100 (JASCO, Easton, MD, USA). [α]D values were obtained from a digital polarimeter (JASCO). Ultraviolet spectra were recorded on a Shimadzu UV-1650PC spectrometer (Shimadzu, Kyoto, Japan). HRESIMS data were obtained on a Shimadzu hybrid ion-trap time-of-flight mass spectrometer (Shimadzu). HPLC was performed with a column (250 mm × 4.6 mm i.d., 5 μm; YMC-Pack-ODS-A), with a PrimeLine Binary pump with RI-101 Shodex refractive index detector and M 525 variable UV detector (Shoko Scientific Co., Yokohama, Japan). Dextrose and agar were purchased from Junsei Chemical Co. Ltd. (Tokyo, Japan), and tryptone, yeast extract, and beef extracts were purchased from Becton, Dickinson and Co. (Sparks, MD, USA). All experimental chemicals were obtained from Duksan Pure Chemicals (Ansan, South Korea). For antimicrobial tests, type strains were collected from Korean Collection for Type Cultures (KCTC, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea). Taxonomy of the Bacterial Strain. During an expedition in 2010, a sediment sample was collected from Gageocho reef, Republic of Korea, and the producing strain designated as 109GGC020 was isolated from the sample by serial dilution technique. The 16S rRNA B
dx.doi.org/10.1021/jf502436r | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
C
3-OH acid
Leu3
Glu
Leu2
Leu1
units
6 7 8 9 10 11
3 4 5
4 NH COOH OCH3 1 2 3 4 5 6 NH 1 2
1 2 3 4 5 6 NH 1 2 3 4 5 6 NH 1 2 3
no.
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)a
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)a
1
dd (8.5, 14.0) dd (4.5, 14.5) m m m m brs
dd (8.5, 6.0, α) m (β) m (γ) m (δ) m (δ) d (6.5)a
1.29 brs
2.32 2.46 3.98 1.48 1.44 1.31 1.29
4.27 1.66 1.66 0.95 0.95 8.27
4.33 dd (9.0, 6.0, α) 1.95 m (β) 2.09 m (β) 2.41 m (γ) 8.23 d (6.5)a 176.7
4.41 1.66 1.66 0.95 0.95 8.13
4.44 1.66 1.66 0.95 0.95 8.00
δH δC
30.8
30.6−30.9
70.0 38.5 26.9
175.0 44.7
3.66 s 174.2 54.0 41.4 26.1 22.2 23.8
31.5
174.8 54.6 28.2
174.8 53.3 41.6 26.1 22.0 23.7
174.3 52.4 41.4 26.1 21.6 23.6
Table 1. 1H and 13C NMR Data of 1−4 in CD3OD
m (α) m (β) m (γ) m (δ) m (δ) d (8.0)a
dd (8.5, 5.5, α) m (β) m (γ) m (δ) m (δ) d (8.0)a
2
dd (14.0, 8.5) dd (10.0, 4.0) m m m m brs
m (α) m (β) m (γ) m (δ) m (δ) d (7.0)a
1.29 m
2.32 2.46 3.97 1.51 1.48 1.35 1.29
4.43 1.66 1.66 0.94 0.94 8.19
4.26 dd (9.0, 6.0, α) 1.95 m (β) 2.10 m (β) 2.45 m (γ) 8.23 d (7.0)a 175.1 52.4
4.40 1.66 1.66 0.94 0.94 8.11
4.33 1.66 1.66 0.94 0.94 7.89
δH
33.2
30.6−30.9
70.0 38.5 26.9
174.9 44.7
174.8 53.2 41.4 26.0 21.6 23.6
31.3
174.1 54.4 28.0
174.7 52.5 41.6 26.1 22.0 23.7
175.1 54.0 41.8 26.1 22.2 23.8
δC
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)
3
2.32 2.46 3.98 1.48 1.47 1.35 1.30 1.17 1.52 1.30 1.30 1.13
4.27 1.65 1.65 0.94 0.94 8.27
dd (14.0, 8.5) dd (14.5, 4.5) m m m m brs m m brs brs m
dd (8.5, 6.0, α) m (β) m (γ) m (δ) m (δ) d (6.5)a
4.33 dd (9.0, 6.0, α) 1.95 m (β) 2.09 m (β) 2.41 m (γ) 8.23 d (6.5)a 176.7
4.42 1.65 1.65 0.94 0.94 8.12
4.45 1.65 1.65 0.94 0.94 7.97
δH
30.6−31.3 40.4 29.3 30.8 35.8 37.9
70.0 38.5 26.9
174.9 44.7
174.2 54.0 41.7 26.1 22.2 23.8
31.5
174.8 54.6 28.1
174.8 53.2 41.6 26.1 22.0 23.6
176.2 52.4 41.4 26.0 21.6 23.5
δC
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)a
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)a
4
2.43 2.31 3.96 1.46 1.45 1.33 1.28 1.28 1.15 1.49 1.28 1.28
4.26 1.63 1.63 0.92 0.92 8.23
dd (4.5, 14.5) dd (8.5, 14.0) m m m m brs brs m m brs brs
dd (8.5, 6.0, α) m (β) m (γ) m (δ) m (δ) d (6.5)a
4.30 dd (9.0, 6.0, α) 1.92 m (β) 2.06 m (β) 2.39 t (4.5, γ) 8.20 d (6.5)a 176.8
4.39 1.63 1.63 0.92 0.92 8.09
4.42 1.63 1.63 0.92 0.92 7.93
δH
δC
30.7−31.2 30.7−31.2 40.4 29.3 30.9 35.8
70.0 38.4 26.9
174.9 44.7
174.2 54.0 41.7 26.1 22.2 23.8
31.4
174.8 54.6 28.3
174.8 53.2 41.6 26.1 22.0 23.6
176.3 52.4 41.3 26.0 21.6 23.5
Journal of Agricultural and Food Chemistry Article
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h in sterile distilled water. The motility bioassay was carried out with the zoospores. A series of solutions for gegeopeptides were prepared in aqueous dimethyl sulfoxide (DMSO). The final concentration of DMSO in the zoospore suspension was less than 1% (v/v), a condition that does not affect the motility of zoospores. The bioassay for the motility inhibition and lysis of zoospores was investigated as described previously.25,26 In brief, to a suspension (360 μL) of zoospores (ca.105/mL), 40 μL of sample solution was added and stirred with a glass rod. A 1% amount of aqueous DMSO was used as a control. Under a light microscope (×10 magnification), the time-course changes of motility and lysis of zoospores were evaluated as described previously.27 The bioassay was repeated twice, and each treatment was replicated thrice. The mean value (%) was calculated (±SE, standard error) of the affected spores in each treatment. Minimum inhibitory concentration (MIC) values of individual compounds were converted to micromolar.28 Further Activity Study against Bacteria and Fungi. A standard “broth dilution assay”29 was employed to evaluate the antimicrobial activity of gageopeptides against pathogenic fungi (R. solani, Botrytis cinerea (B. cinerea), and Colletotrichum acutatum (C. acutatum)), Gram positive bacteria (B. subtilis and Staphylococcus aureus (S. aureus)) and Gram negative bacteria (Salmonella typhi (S. typhi) and Pseudomonas aeruginosa (P. aeruginosa)). Briefly, a series of solutions for gegeopeptides were prepared over the range of 0.5−256 μg/mL in 96-well plates. Type strains were cultured overnight, and the final concentration (1.5 × 108 cfu/mL) of each strain in the culture broth was adjusted by comparing the culture turbidity with the 0.5 McFarland Standard. To each solution of gageopeptides 1−4, 30 μL of culture broth was added and the final volume was adjusted to 200 μL using the respective fresh culture medium. The plates were incubated for 24 h at 37 °C for bacteria and 48 h at 30 °C for fungi.28 The minimum inhibitory concentration (MIC, the lowest concentration of an antimicrobial compound that inhibits the visible growth of a microorganism) was recorded and expressed in micromolar in Result and Discussion.28 Cytotoxicity by SRB Assay. Cancer cell growth inhibitory activity of gageopeptides 1−4 was carried out according to a sulforhodamine B (SRB) assay.30 The selected cancer cell lines (brest cancer, MDA-MB231; colon cancer, HCT-15; prostate cancer, PC-3; lung cancer, NCIH23; stomach cancer, NUGC-3; and renal cancer, ACHN) were added into 96-well plates. A series of solutions of gageopeptides with the concentrations of 6.25−50 μM were prepared and added to the plates. The plates were incubated for 48 h, and anchorage-dependent cells were fixed with 50% (w/v) trichloroacetic acid and stained for 60 min. The SRB solution (0.4% sulforhodamine B in 1% acetic acid) was used to wash out excess dye. The protein-bound dye was dissolved in 10 mM Tris base solution for optical density determination at 510 nm using a microplate reader. Using graphed prism software, the GI50 values of gageopeptides were calculated. Cytotoxicity Test by WST-1 Assay. Inhibition of cancer cell growth for compounds 1−4 was determined by WST-1 (4-[3−4iodophenyl]-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene disulfonate) assay.31,32 In brief, the human myeloid leukemia K-562 cells were maintained in RPMI 1640 medium in 5% fetal bovine serum, supplemented with humidified air containing 5% CO2 at 37 °C. Compounds 1−4 were added to the properly distributed cells (2 × 104 cells/mL) in 96-well plates. The plates were incubated for 5 days under the preceding condition. A 50 μL aliquot of WST-1 stock solution (1.0 mg/mL) was added to the suspensions, and the plates were further incubated for 4 h. After aspirating off the supernatant with a microplate washer, the formazen crystals were dissolved by adding 150 μL of DMSO in each well. Finally, the absorbance was measured using a microplate reader at 480 nm, and IC50 values were calculated. Cytotoxicity Test by MTT Assay. The MTT assay was carried out to determine cancer cell viability.33 In brief, the RAW 264.7 cells (mouse leukemic monocyte macrophage cell line, 5 × 104 cells/mL), were cultured in a 96-well plate at 37 °C, and treated with varying concentrations (6.25, 12.5, 25, and 50 μM) of gageopeptides 1−4 for 24 h. Cells treated only with medium were used as a negative control group. The supernatant of each well was removed, and 20 μL of MTT
11.9 19.8 174.9
37.9
Article
19.8 23.2
11.9
0.87 m 0.87 m 14.6 23.5
30.6
0.89, t (7.5) 0.91, m 14.6 23.5
33.2
0.87 t 0.87 m 13 14 15
H NMR data recorded in CD3OH. a1
1.29 brs 12
no. units
Table 1. continued
δH
1
δC
1.29 m
δH
2
δC
1.30 brs 0.87 m
δH
3
δC
1.11 1.28 0.87 0.87 0.87
m brs m m m
δH
4
δC
Journal of Agricultural and Food Chemistry
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solution (5 mg/mL in phosphate buffered saline) and 100 μL of medium were added into the plates. The plates were incubated for another 4 h, and the formazen crystals were dissolved in DMSO (100 μL). The absorbance at 540 nm was measured using a microplate reader. The relative cell viability (%) was measured as a percentage relative to the untreated control cells.
acid residues. An additional oxygenated proton resonating at δH 3.98 displaying TOCSY and HMBC correlations with the carbonyl carbon at δC 175.0 and an aliphatic chain confirmed the presence of a fatty acid unit (e) in 1. The 13C NMR spectrum of 1 contained eight methyl carbons; six of them were found to constitute three Leu residues, and therefore, the remaining two methyls should be present in the fatty acid unit. The carbon resonating at δC 14.6 was assigned as a terminal methyl, and the methyl signal resonating at δC 23.5 was found to form a side chain with the fatty acid. By the detailed analysis of TOCSY and HMBC data, the position of the methyl group (δC 23.5) was determined to be at C-11 in the fatty acid chain. Judging from its molecular weight, the fatty acid was primarily assigned to be a C14 fatty acid connected to the N-terminal amine of the peptide. The LC-MS (m/z 243.01 [M − H]−) analysis of the acid hydrolysate of 1 confirmed its chain length. Thus, the fatty acid was assigned as a 3-β-hydroxy-11methyltridecanoic acid.20,21 Finally, the structure of 1 was determined to be Leu1-Leu2-Glu-Leu3-3-β-hydroxy-11-methyltridecanoic acid by HMBC and ROESY experiments (Figure 1). The molecular formula of 2 was determined to be C39H72N4O9 according to the HRESIMS result. The 1H and 13 C NMR data displayed a close similarity between 1 and 2 (Table 1), indicating the lipopeptidic nature of 2. However, the presence of an additional oxygenated methyl peak at δH 3.66 and the molecular weight deference of 2 (C2H4 more) from 1 prompted us to conduct a detailed investigation of its structure. Therefore, a series of 1D and 2D NMR experiments were performed to clarify the structure of 2. These NMR data analyses revealed the similar peptidic structure of 2, consisting of Leu and Glu residues, but the methoxy proton at δH 3.66 did not show any correlations in both COSY and TOCSY spectra. However, HMBC correlations between this proton and a carboxylic carbon at δC 176.7 were found, indicating the presence of a OMeGlu unit in 2. Based on the HMBC and ROESY spectra analysis, the sequence of these amino acid residues in the peptide moiety was assigned as Leu1-Leu2-Leu3OMeGlu. Similar to 1, a 3-β-hydroxy fatty acid was also determined from the observation of correlations of the oxygenated methine proton at δH 3.97 with the methylene protons at δH 2.32/2.46 in the COSY and TOCSY spectra, as well as with a carbonyl carbon at δC 174.9 in the HMBC spectrum. Initially, the elemental composition and molecular formula of 2 suggested that the fatty acid had a molecular formula of C15H30O3. Additional LC-MS results (m/z at 257.08 [M − H]−) of the fatty acid obtained from the acid hydrolysis of 2 confirmed the molecular formula. Thus, the fatty acid was determined as a 3-β-hydroxy-12-methyltetradecanoic acid. Finally, the structure of lipotetrapeptide 2 was constructed as Leu1-Leu2-Leu3-OMeGlu-3-β-hydroxy-12-methyltetradecanoic acid, based on the extensive 2D NMR data analysis (Figure 1). The molecular formula of 3 was assigned to be C37H68N4O9 by means of the HRESIMS result, which was the same as that of 1. Initially, the 1H NMR data of 3 were found very similar to 1. The difference between compounds 1 and 3 was the presence of several carbon resonances in the upfield region in the 13C NMR data of 3. The detailed analysis of a complete set of 1D and 2D NMR data of 3 indicated the presence of the same amino acid residues with the same sequence as 1. Moreover, the primary investigation on the molecular weight of 3 and further LC-MS analysis of the hydrolysate of 3 revealed that the molecular formula of the fatty acid in 3 was the same as
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RESULTS AND DISCUSSION The molecular formula of 1 was found to be C37H68N4O9 based on the HRESIMS data analysis. The 1H NMR data (recorded in both CD3OD and CD3OH) analysis of pure compound 1 revealed its lipopeptidic nature (Table 1), showing amide proton signals at δH 7.99−8.28, α-methine groups at δH 4.26− 4.44, a broad singlet at 1.29, and methyl signals at δH 0.88− 0.97. This initial prediction of the lipopeptidic nature of 1 was further confirmed by the observation of carbonyl/carboxylic carbons at δC 174.2−176.7 and a group of CH2 carbons at δC 30.6−30.9 in the 13C NMR spectrum of 1, which were attributable to amino acid residues and an aliphatic chain, respectively. Later, the detailed interpretation of NMR data revealed five partial structures a−e, corresponding to four amino acid residues and a lipid moiety that constitute 1 (Figure 2). The glutamic acid unit (substructure c) was elucidated from
Figure 2. Assignment of partial structures by COSY and TOCSY correlations and complete structure with key HMBC and ROESY correlations of 1.
the TOCSY correlations of the amide proton at δH 8.23 with an α-methine proton at δH 4.33 and the methylene signals at δH 1.95/2.09, and the α-methine proton also showed TOCSY correlations with the methylenes at δH 1.95/2.09 and 2.41. Furthermore, HMBC correlations were observed between the methylene signals at δH 1.95/2.09 and 2.41 and a carboxylic carbon resonating at δC 176.7, and between the protons at δH 1.95/2.09 and a carbonyl carbon at δC 174.8, confirming the glutamic acid residue in 1. In a similar way, the substructures a−e were assigned by the analysis of 1D and 2D NMR data (Figure 2). The sequence of these amino acid residues was established as Leu1-Leu2-Glu-Leu3 by the detailed interpretation of ROESY and HMBC correlations between α-methines, NH protons, and amide carbonyl carbons of respective amino E
dx.doi.org/10.1021/jf502436r | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Figure 3. Time-course motility inhibitory and lytic activities of (A) compound 1 and (B) compound 2 against zoospores of the late blight pathogen P. capsici at 0.02 μM (investigations were performed in triplicate).
Table 2. Inhibitory and Lytic Activities of Compounds 1 and 2 against P. capsici motility inhibitory and zoosporicidal activity over control (% ± SE)a 15 min dose (μM)
1
2
0.02 0.04 0.07 0.09 0.02 0.04 0.06 0.09 0.10
inhibited motility 74 89 100 100 0 20 69 84 100
± ± ± ± ± ± ± ± ±
whi6 8 0 0 0 3 6 7 0
30 min lysed 49 57 99 100 0 0 0 0 0
± ± ± ± ± ± ± ± ±
5 4 1 0 0 0 0 0 0
45 min
60 min
inhibited motility
lysed
inhibited motility
lysed
inhibited motility
lysed
87 ± 8 100 ± 0 100 ± 0 nt 41 ± 5 70 ± 8 82 ± 9 98 ± 2 100 ± 0
67 ± 6 92 ± 5 100 ± 0 nt 0±0 0±0 14 ± 5 25 ± 9 34 ± 4
92 ± 5 100 ± 0 nt nt 55 ± 6 88 ± 8 91 ± 9 98 ± 2 100 ± 0
79 ± 9 100 ± 0 nt nt 0±0 10 ± 4 21 ± 6 33 ± 7 49 ± 3
98 ± 2 nt nt nt 78 ± 6 92 ± 10 98 ± 3 100 ± 0 100 ± 0
86 ± 7 nt nt nt 0±0 21 ± 5 26 ± 5 45 ± 7 65 ± 4
Data presented here are the average value ± SE of at least three replications in each dose of compounds. nt, not tested. Compounds 3 and 4 did not show any activity against zoospores of P. capsici under the conditions tested.
a
Figure 4. Minimum inhibitory concentrations (μM) of 1−4 against (A) Gram positive and Gram negative bacteria and (B) fungi.
that of 1. However, the presence of additional methine carbons at δC 37.9 and 40.4 and methyl carbons at δC 11.9 and 19.8 in the 13C NMR spectrum indicated that 3 had a different fatty acid from 1. By the detailed interpretation of HMBC and TOCSY correlations, these additional methyl carbons were found to be located at C-9 and C-11 positions, and the fatty acid was assigned as a 3-β-hydroxy-9,11-dimethyltridecanoic acid.20 Finally, the structure of 3 was determined as Leu1-Leu2Glu-Leu3-3-β-hydroxy-9,11-dimethyltridecanoic acid by the extensive interpretation of 2D NMR data (Figure 1). The molecular formula of 4 was deduced to be C38H70N4O9 based on the HRESIMS data, and the 1H and 13C NMR data indicated that 4 was an additional analogue of 3. But, it was found that compound 4 had a molecular weight 14 Da more than 3, indicating the presence of an additional methylene group that could be present either in the fatty acid chain or in the peptide part. The careful interpretation of the NMR data
revealed that compounds 3 and 4 had the same peptide moiety, suggesting the additional CH2 group should be located in the fatty acid unit. This fact was further confirmed by the LC-MS analysis of the hexane phase of the acid hydrolysate of 4. Thus, the molecular formula of the fatty acid in 4 was determined to be C15H30O3. The fatty acid unit was found to possess same side chain as 3 with an additional methylene group and determined as a 3-β-hydroxy-9,11-dimethyltetradecanoic acid by 2D data analysis.21 Thus, the final structure of 4 was assigned as Leu1-Leu2-Glu-Leu3-3-β-hydroxy-9,11-dimethyltetradecanoic acid by HMBC and ROESY correlations (Figure 1). The absolute stereochemistry of the amino acid residues in 1−4 was found to be L-form by Marfey’s method. In addition, the absolute stereochemistry at C-3 of the fatty acid in 1 was assigned to be R by Mosher’s MTPA method.34 The absolute configuration of 3-hydroxy fatty acids in 2−4 (obtained from F
dx.doi.org/10.1021/jf502436r | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry the hydrolysis of 2−4 followed by extraction with hexane) was also found to be R by the comparison of their optical rotation values {[α]27D −22 (c o.1, MeOH), [α]27D −45 (c o.1, MeOH), and [α]27D −24 (c o.1, MeOH), respectively} with the optical rotation value [α]27D −26 (c o.1, MeOH) of the 3-hydroxy fatty acid in 1, as well as literature reviews, as the (R)-3-hydroxy fatty acid has the negative [α]D value in MeOH.20,21,35,36 Compounds 1−4 were tested against both bacteria and fungi. These compounds were found to inhibit the growth of these microorganisms. In particular, compounds 1−4 displayed significant activity against R. solani and P. capsici. Compound 1 showed both inhibitory and lytic activity against P. capsici at a concentration of 0.02 μM, whereas compound 2 showed only inhibitory activity at the same concentration (Figure 3). However, compound 2 exhibited lytic activities against P. capsici when the concentration was increased (Table 2). It may be noteworthy that compound 2 had an OMeGlu amino acid residue in the peptide unit and an additional CH2 group in the fatty acid chain compared to 1. It was also revealed that compounds 1 and 2 exhibited stronger antifungal activity than 3 and 4 (Figure 4B). Compounds 1 and 2 were structurally different from compounds 3 and 4 in terms of having less side chains in the fatty acid units, suggesting that the side chain length and the additional methyl group in the aliphatic chain are important for antifungal activity. Usually, the fungal cell wall consists of three main components, chitin, glucans, and proteins. Antifungal agents display their activity by disrupting the integrity of this membrane or inhibiting sterol synthesis. Conversely, lipopeptides inhibit the synthesis of β-1,3-D-glucan in the cell wall. In fact, without β-1,3-D-glucan, the integrity of the fungal cell wall is compromised, leading to increased osmotic fragility and the eventual lysis of the fungal cell.20,37 Since compounds 1−4 were found to exhibit potent antimicrobial activities, we became curious to know their possible cytotoxic effects. Accordingly, compounds 1−4 were tested against a panel of six human cancer cell lines according to a sulforhodamine B (SRB) assay. Interestingly, these compounds did not exhibit any activity (GI50 > 25 μM) against these cancer cell lines. The noncyotoxic character of these compounds was further checked against the leukemia cell line using WST-1 and MTT assays. From these detailed bioactivity results, it may be concluded that these lipotetrapeptides would be useful materials to develop environmentally friendly nontoxic antifungal agents.
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ACKNOWLEDGMENTS
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REFERENCES
This research was supported in part by the Korea Institute of Ocean Science and Technology (Grant PE99273 to H.J.S.).
(1) Anderson, N. A. The genetics and pathology of Rhizoctonia solani. Ann. Rev. Phytopathol. 1982, 20, 329−347. (2) El Bakali, M. A.; Martin, M. P.; Garcia, F.; Moret, A.; Nadal, M.; Monton, C. First report of Rhizoctonia solani AG-3 in Catalonia (NE Spain). Plant Dis. 2000, 84, 806. (3) El Bakali, M. A.; Martin, M. P.; Garcia, F.; Moret, A.; Nadal, M. Identification of Spanish isolates of Rhizoctonia solani from potato by anastomosis grouping, ITS-RFLP and RAMS-fingerprinting. Phytopathol. Med. 2003, 42, 167−176. (4) Marin, J. P.; Segarra, J.; Almacellas, J. Enfermedades de los cereales en Cataluña en 1988−90. Invest Agrar.: Prod. Prot. Veg. 1992, 7, 261−275. (5) Melero-Vara, J. M.; Jimenez-Diaz, R. M. Etiology, incidence and distribution of cotton seedling damping-off in southern Spain. Plant Dis. 1990, 74, 597−600. (6) Akgül, D. S.; Mirik, M. Biocontrol of Phytophthora capsici on pepper plants by Bacillus megaterium strain. J. Plant Pathol. 2008, 90, 29−34. (7) Strange, R. N.; Scott, P. R. Plant disease: A threat to global food security. Ann. Rev. Phytopathol. 2005, 43, 83−116. (8) Suprapta, D. N. Potential of microbial antagonists as bio-control agents against plant pathogens. J. ISSAAS 2012, 18, 1−8. (9) Nannipieri, P. The potential use of soil enzymes as indicators of productivity, sustainability and pollution. In Soil biota-management in sustainable farming systems; Pankhurst, C. E., Doube, B. M., Gupta, V. V. S. R., Grace, P. R., Eds.; CSIRO: Collingwood, Australia, 1994; pp 33−140. (10) Wage workers in agriculture: condition of employment and work; sectoral activities program. Report for discussion at the tripartite meeting on improving conditions of employment and work of agricultural wage workers in the context of economic restructuring. International Labor Organization: Geneva, Switzerland, 1996. (11) Miller, G. T. Sustaining the Earth, 6th ed.; Thompson Learning: Pacific Grove, CA, USA, 2004; Chapter 9, pp 211−216. (12) Singh, P.; Cameotra, S. S. Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol. 2004, 22, 142− 146. (13) Cho, S. J.; Hong, S. Y.; Kim, J. Y.; Park, S. R.; Kim, M. K.; Lim, W. J. Endophytic Bacillus sp. CY22 from a balloon flower (Platycodon grandif lorum) produces surfactin isoforms. J. Microbiol. Biotechnol. 2003, 13, 859−865. (14) Cao, X.; Wang, A. H.; Jiao, R. Z.; Wang, C. L.; Mao, D. Z.; Yan, L. Surfactin induces apoptosis and G2/M arrest in human breast cancer MCF-7 cells through cell cycle factor regulation. Cell Biochem. Biophys. 2009, 55, 163−171. (15) Cao, X. H.; Wang, A. H.; Wang, C. I.; Mao, D. Z.; Lu, M. F.; Cui, Y. Q. Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway. Chem.-Biol. Interact. 2010, 183, 357−362. (16) Vollenbroich, D.; Ozel, M.; Vater, J.; Kamp, R. M.; Pauli, G. Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis. Biologicals 1997, 25, 289−297. (17) Kim, S. D.; Cho, J. Y.; Park, H. J.; Lim, C. R.; Lim, J. H.; Yun, H. I. A comparison of the anti-inflammatory activity of surfactin A, B, C, and D from Bacillus subtilis. J. Microbiol. Biotechnol. 2006, 16, 1659. (18) Park, S. Y.; Kim, Y. H. Surfactin inhibits immunostimulatory function of macrophages through blocking NK-jB, MAPK and Akt pathway. Int. Immunopharmacol. 2009, 9, 886−893. (19) Kurtz, M. B.; Douglas, C. M. Lipopeptide inhibitors of fungal glucan synthase. J. Med. Vet. Mycol. 1997, 35, 79−86. (20) Tareq, F. S.; Lee, M.; Lee, H. S.; Lee, Y. J.; Lee, J. S.; Hasan, C. M.; Islam, M. T.; Shin, H. J. Gageotetrins A−C, noncytotoxic
ASSOCIATED CONTENT
S Supporting Information *
Figures showing 1H, 13C, and 2D NMR spectra of 1−4, HPLC comparison of amino acid residues in 1 with Marfey’s derivatives of standard L- and D-amino acids, LC-MS data for 3 and 4. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel.: +82-31-400-6172. Fax: +82-31400-6170. Notes
The authors declare no competing financial interest. G
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antimicrobial linear lipopeptides from a marine bacterium Bacillus subtilis. Org. Lett. 2014, 16, 928−93. (21) Tareq, F. S.; Lee, M.; Lee, H. S.; Lee, Y. J.; Lee, J. S.; Shin, H. J. Gageostatins A−C, antimicrobial linear lipopeptides from a marine Bacillus subtilis. Mar. Drugs 2014, 12, 871−885. (22) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. High-field FT NMR application of Mosher’s method. The absolute configuration of marine terpenoids. J. Am. Chem. Soc. 1991, 113, 4092−4096. (23) Marfey, P. Determination of D-amino acids. II. Use of a bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. Carlsberg Res. Commun. 1984, 49, 591−596. (24) Erwin, D. C.; Ribeiro, O. K. Phytophthora Diseases Worldwide; APS Press: St. Paul, MN, USA, 1996; 562 pp. (25) Islam, M. T.; Hashidoko, Y.; Deora, A.; Ito, T.; Tahara, S. Suppression of damping-off disease in host plants by the rhizoplane bacterium Lysobacter sp. strain SB-K88 is linked to plant colonization and antibiosis against soilborne Peronosporomycetes. Appl. Environ. Microbiol. 2005, 71, 3786−3796. (26) Islam, M. T.; Von, T. A.; Laatsch, H. Protein kinase C is likely to be involved in zoosporogenesis and maintenance of flagellar motility in the peronosporomycete zoospores. Mol. Plant−Microbe Interact. 2011, 24, 938−947. (27) Islam, M. T.; Ito, T.; Sakasal, M.; Tahara, S. Zoosporicidal activity of polyflavonoid tannin identified in Lannea coromandelica stem bark against phytopathogenic oomycete Aphanomyces cochlioides. J. Agric. Food Chem. 2002, 50, 6697−6703. (28) Tareq, F. S.; Kim, J. H.; Lee, M. A.; Lee, H. S.; Lee, J. S.; Lee, Y. J.; Shin, H. J. Antimicrobial gageomacrolactins characterized from the fermentation of the marine-derived bacterium Bacillus subtilis under optimum growth conditions. J. Agric. Food Chem. 2013, 61, 3428− 3434. (29) Appendino, G.; Gibbons, S.; Giana, A.; Pagani, A.; Grassi, G.; Stavri, M.; Smith, E.; Rahman, M. M. Antibacterial cannabinoids from Cannabis sativa: a structure-activity study. J. Nat. Prod. 2008, 71, 1427−1430. (30) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMohan, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyed, M. R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 1990, 82, 1107−1112. (31) Seo, Y.; Cho, K. W.; Lee, H. S.; Rho, J. R.; Shin, J. New acetylenic enol ethers of glycerol from the sponge Petrosia sp. J. Nat. Prod. 1999, 62, 122−126. (32) Chucheep, K.; Kanlayanarat, S.; Maneerat, C.; Matsuo, T. Application of WST-1 to measurement of cell viability in low temperature-stressed explants of tropical vegetables. J. Food Agric. Environ. 2005, 3, 262−268. (33) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (34) Freire, F.; Seco, J. M.; Emilio, Q.; Riguera, R. Determining the absolute stereochemistry of secondary/secondary diols by 1H NMR: basis and applications. J. Org. Chem. 2005, 70, 3778−3790. (35) Jenske, R.; Vetter, W. Enantioselective analysis of 2- and 3hydroxy fatty acids in food samples. J. Agric. Food Chem. 2008, 56, 11578−11583. (36) Maider, P.; Francoise, P.; Jean, W. Solid-phase synthesis of surfactin, a powerful biosurfactant produced by Bacillus subtilis, and of four analogues. Int. J. Pept. Res. Ther. 2005, 11, 195−202. (37) Kartsonis, N. A.; Nielsen, J.; Douglas, C. M. Caspofungin: the first in a new class of antifungal agents. Drug Resist. Updates 2003, 6, 197−218.
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Non-cytotoxic Antifungal Agents: Isolation and Structures of Gageopeptides A−D from a Bacillus Strain 109GGC020 Fakir Shahidullah Tareq,†,‡ Min Ah Lee,‡ Hyi-Seung Lee,‡ Yeon-Ju Lee,‡ Jong Seok Lee,‡ Choudhury M. Hasan,§ Md. Tofazzal Islam,∥ and Hee Jae Shin*,†,‡ †
Department of Marine Biotechnology, Korea University of Science and Technology, Daejeon 350-360, Republic of Korea Marine Natural Products Laboratory, Korea Institute of Ocean Science and Technology, Ansan 426-744, Republic of Korea § Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka 1000, Bangladesh ∥ Department of Biotechnology, Bangabandhu Sheikh Muzibur Rahman Agricultural University, Dhaka 1706, Bangladesh ‡
S Supporting Information *
ABSTRACT: Antifungal resistance and toxicity problems of conventional fungicides highlighted the requirement of search for new safe antifungal agents. To comply with the requirement, we discovered four new non-cytotoxic lipopeptides, gageopeptides A−D, 1−4, from a marine-derived bacterium Bacillus subtilis. The structures and stereochemistry of gageopeptides were determined by NMR data analysis and chemical means. Gageopeptides exhibited significant antifungal activities against pathogenic fungi Rhizoctonia solani, Botrytis cinerea, and Colletotrichum acutatum with minimum inhibitory concentration (MIC) values of 0.02−0.06 μM. In addition, these lipopeptides showed significant motility inhibition and lytic activities against zoospores of the late blight pathogen Phytophthora capsici. These compounds also showed potent antimicrobial activity against Gram positive and Gram negative bacteria with MIC values of 0.04−0.08 μM. However, gageopeptides A−D did not exhibit any cytotoxicity (GI50 > 25 μM) against cancer cell lines in sulforhodamine B (SRB), 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT), and WST-1 ((4-[3−4-iodophenyl]-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene disulfonate)) assays, demonstrating that these compounds could be promising candidates for the development of non-cytotoxic antifungal agents. KEYWORDS: Bacillus subtilis, gageopeptides, antifungal agents, non-cytotoxicity
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INTRODUCTION Fungal diseases are very common crop-devastating diseases in agricultural practice. For example, Rhizoctonia solani (R. solani) is a plant pathogenic fungus with a wide host range and worldwide distribution. This pathogen is very destructive to many crops, including agronomic, ornamental, and forestry species.1 This species has been reported to enormously reduce the growth and quality of crops such as cereals, potato, lettuce, and cotton in Spain.2−5 Phytophthora capsici (P. capsici), another economically important pathogen, causes extensive damage in cucumber, pepper, tomato, and beans. It is well recognized to cause crown, leaf, and fruit blight, wilting of the whole plant, and dark purplish discoloration of the stem.6 Similarly, a diverse group of fungi are recognized to cause severe plant diseases, resulting in a significant loss in agricultural practice.7 Therefore, it is very important to control plant diseases to secure the level of yield both quantitatively and qualitatively. Farmers often heavily rely on the application of commercial fungicides to secure the quality and production of crops. Most of the fungicides are not compatible with the environment or health; their excessive application and misuse can also cause acute and chronic toxicity.8,9 According to a review by the International Labor Organization (ILO), about 14% of all occupational injuries were caused by exposure to synthetic pesticides and other agrochemicals.10 Moreover, the World Health Organization (WHO) and the United Nations Environ© XXXX American Chemical Society
ment Programme (UNEP) reported that three million farmers in the developing countries suffer from severe poisoning by commercial pesticides, resulting in the deaths of about 18,000 of them each year.11 However, the environmental problems caused by synthetic fungicides have brought a significant change to people who work in the agricultural field toward the use of harmful pesticides. Therefore, it has now become an urgent issue to develop environmentally friendly alternative pesticides to synthetic chemicals to protect plants from diseases. Lipopeptides produced nonribosomally by bacteria and fungi have been reported to be biodegradable and less toxic, and have various biomedical applications.12 As illustrations, lipopeptides of surfactins, iturins, mycosubtilin, plipastatin, halobacillin, fengycin, and echinocandin families produced by bacterial origins have recently received much attention due to their potent biological activities, including antimicrobial, antitumor, antiviral, anti-inflammatory, and immunosuppressive activities.13−18 In particular, echinocandins and pneumocandins are now approved antifungal drugs which inhibit the synthesis of glucan in the cell wall, via noncompetitive inhibition of the enzyme 1,3-β-D-glucan synthase in pathogenic fungi including Received: February 13, 2014 Revised: May 25, 2014 Accepted: May 26, 2014
A
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Aspergillus and Candida.19 In our previous studies,20,21 we have discovered three novel lipotetrapeptides, gageotetrins A−C and surfactin like heptapeptides, gageostatins A−C with significant antimicrobial properties. Further investigations on the ethyl acetate (EtOAc) extract obtained from the same bacterium Bacillus subtilis (B. subtillis) afforded four new lipopeptides, gageopeptides A−D, 1−4 (Figure 1), which displayed
sequence of the strain 109GGC020 was submitted to GenBank under accession number JQ927413. A BLAST search result showed the sequence of 109GGC020 to be 100% similar to B. subtilis. Isolation of Compounds 1−4. Batch fermentation (60 L) of the strain B. subtilis 109GGC020 was carried out in 2 L flasks. For 1 L, the medium composition (w/v) was 1% dextrose, 0.1% yeast extract, 0.1% beef extract, 0.2% tryptone, 100% natural seawater, and pH 7.1. The culture broth was separated from the cells by centrifugation after 7 days of cultivation at 28 °C. The broth was extracted with EtOAc and the solvent was removed under reduced pressure at 40 °C to dryness. The EtOAc extract (11.8 g) was fractionated by an ODS open column chromatography using methanol:water (1:4, 2:3, 3:2, 4:1, and 100:0; v/v) as eluent. The 100% MeOH fraction was refractionated and purified by ODS semipreparative and analytical HPLCs with a solvent system of 95% MeOH in H2O to yield 1−4. Gageopeptide A (1; 4.6 mg; 0.04%): amorphous solid; [α]27D −21 (c 0.1, MeOH). IR (MeOH; νmax, cm−1): 3285 (NH), 1620−1689 (CO), 2923 (aliphatic chain). 1H and 13C NMR data (CD3OD): Table 1. HRESIMS (m/z): 735.4883 [M + Na]+. Calcd for C37H68N4O9: 735.4884 [M + Na]+; Gageopeptide B (2; 1.8 mg; 0.02%): amorphous solid; [α]27D −40 (c 0.1, MeOH). IR (MeOH; νmax, cm−1): 3278 (NH), 1642 (CO), 2927 (aliphatic chain). 1H and 13C NMR data (CD3OD): Table 1. HRESIMS (m/z): 763.5192 [M + Na]+. Calcd for C39H72N4O9: 763.5197 [M + Na]+. Gageopeptide C (3; 5.9 mg; 0.05%): amorphous solid; [α]27D −20 (c 0.1, MeOH). IR (MeOH; νmax, cm−1) 3373 (NH), 1625 (CO), 2970 (aliphatic chain). 1H and 13C NMR data (CD3OD): Table 1. HRESIMS (m/z): 735.4883 [M + Na]+. Calcd. for C37H68N4O9: 735.4884 [M + Na]+. Gageopeptide D (4; 7.8 mg; 0.07%): amorphous solid; [α]27D −70 (c 0.05, MeOH). IR (MeOH; νmax, cm−1): 3287 (NH), 1629 (CO), 2959 (aliphatic chain). 1H and 13C NMR data (CD3OD): Table 1. HRESIMS (m/z): 749.5044 [M + Na]+. Calcd for C38H70N4O9: 749.5040 [M + Na]+. Determination of Absolute Stereochemistry of 1−4. The absolute stereochemistry of amino acid residues and at the stereocenter C-3 of 3-hydroxy fatty acids in 1−4 was determined by using the methods described previously.20,21 In brief, compound 1 (1.9 mg) was hydrolyzed using 6 N HCl (500 μL) at 120 °C for 23 h. The hydrolysate was diluted with water and extracted with hexane. The hexane layer was evaporated by a stream of nitrogen, and the water layer was concentrated to dryness under reduced pressure. To determine the absolute configuration at the stereocenter C-3 of the fatty acid,22 the hexane extract was derivatized with Mosher’s reagent and the aqueous extract was used for the determination of absolute stereochemistry of amino acids by Marfey’s method.23 In an entirely analogous way, compounds 2−4 were hydrolyzed and their absolute stereochemistries were assigned. Fatty acid in 1: 1.5 mg; colorless oil. 1H NMR data (CD3OD): δH 2.34 (H-2a, dd, J = 15.0, 8.5 Hz), 2.43 (H-2b, dd, J = 15.5, 5.0 Hz), 3.96 (H-3, m), 1.33 (H-4a, m), 1.45 (H-4b, m), 1.47 (H2-5, m), 1.29 (H2-6−9, brs), 1.18 (H-10, m), 1.50 (H-11, m, overlapped), 1.29 (H12, m, overlapped), 0.86 (H3-13, t, J = 5.5 Hz), 0.87 (H3-14, d, J = 6.5 Hz). LC-MS (m/z): 243.01 [M − H]−. Optical rotation ([α]27D): −26 (c o.1, MeOH). Activity Study of 1−4 against P. capsici. P. capsici was received from Prof. W. Yuanchao of Nanjing Agricultural University, China. Mycelia of P. capsici were cultured on sterile clarified 10% V8 agar in Petri dishes and incubated at 25 °C in the dark for 4 days.24 The agar culture from the Petri dishes incubated for 1−2 weeks was cut into six pieces, covered with sterile distilled water, and cultured in Petri dishes again at 25 °C in the dark to induce sporangium formation. Every 12 h, the water was replaced by fresh sterile distilled water. In this way, after changing the water 3 times, an abundance of sporangia were induced. The Petri dishes with large numbers of sporangia were incubated at 4 °C for 30 min, and kept at room temperature for another 30 min to induce zoospore release. The movement of sporangia and zoospores was checked on a regular basis under an Olympus light microscope. The zoospores remained motile for 10−12
Figure 1. Structures of compounds 1−4.
significant activity against pathogenic fungi and bacteria. The cytoxicity of gageopeptides against human cancer cell lines was also investigated during the study, and the results demonstrated that these new lipopeptides are potent non-cytotoxic antifungal agents.
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MATERIALS AND METHODS
General Experimental Procedures. One- (1D) and twodimensional (2D) NMR data were recorded on a Varian Unity 500 NMR spectrometer (Varian Inc., Palo Alto, CA, USA). IR spectra were measured on a JASCO FT/IR-4100 (JASCO, Easton, MD, USA). [α]D values were obtained from a digital polarimeter (JASCO). Ultraviolet spectra were recorded on a Shimadzu UV-1650PC spectrometer (Shimadzu, Kyoto, Japan). HRESIMS data were obtained on a Shimadzu hybrid ion-trap time-of-flight mass spectrometer (Shimadzu). HPLC was performed with a column (250 mm × 4.6 mm i.d., 5 μm; YMC-Pack-ODS-A), with a PrimeLine Binary pump with RI-101 Shodex refractive index detector and M 525 variable UV detector (Shoko Scientific Co., Yokohama, Japan). Dextrose and agar were purchased from Junsei Chemical Co. Ltd. (Tokyo, Japan), and tryptone, yeast extract, and beef extracts were purchased from Becton, Dickinson and Co. (Sparks, MD, USA). All experimental chemicals were obtained from Duksan Pure Chemicals (Ansan, South Korea). For antimicrobial tests, type strains were collected from Korean Collection for Type Cultures (KCTC, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, South Korea). Taxonomy of the Bacterial Strain. During an expedition in 2010, a sediment sample was collected from Gageocho reef, Republic of Korea, and the producing strain designated as 109GGC020 was isolated from the sample by serial dilution technique. The 16S rRNA B
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C
3-OH acid
Leu3
Glu
Leu2
Leu1
units
6 7 8 9 10 11
3 4 5
4 NH COOH OCH3 1 2 3 4 5 6 NH 1 2
1 2 3 4 5 6 NH 1 2 3 4 5 6 NH 1 2 3
no.
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)a
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)a
1
dd (8.5, 14.0) dd (4.5, 14.5) m m m m brs
dd (8.5, 6.0, α) m (β) m (γ) m (δ) m (δ) d (6.5)a
1.29 brs
2.32 2.46 3.98 1.48 1.44 1.31 1.29
4.27 1.66 1.66 0.95 0.95 8.27
4.33 dd (9.0, 6.0, α) 1.95 m (β) 2.09 m (β) 2.41 m (γ) 8.23 d (6.5)a 176.7
4.41 1.66 1.66 0.95 0.95 8.13
4.44 1.66 1.66 0.95 0.95 8.00
δH δC
30.8
30.6−30.9
70.0 38.5 26.9
175.0 44.7
3.66 s 174.2 54.0 41.4 26.1 22.2 23.8
31.5
174.8 54.6 28.2
174.8 53.3 41.6 26.1 22.0 23.7
174.3 52.4 41.4 26.1 21.6 23.6
Table 1. 1H and 13C NMR Data of 1−4 in CD3OD
m (α) m (β) m (γ) m (δ) m (δ) d (8.0)a
dd (8.5, 5.5, α) m (β) m (γ) m (δ) m (δ) d (8.0)a
2
dd (14.0, 8.5) dd (10.0, 4.0) m m m m brs
m (α) m (β) m (γ) m (δ) m (δ) d (7.0)a
1.29 m
2.32 2.46 3.97 1.51 1.48 1.35 1.29
4.43 1.66 1.66 0.94 0.94 8.19
4.26 dd (9.0, 6.0, α) 1.95 m (β) 2.10 m (β) 2.45 m (γ) 8.23 d (7.0)a 175.1 52.4
4.40 1.66 1.66 0.94 0.94 8.11
4.33 1.66 1.66 0.94 0.94 7.89
δH
33.2
30.6−30.9
70.0 38.5 26.9
174.9 44.7
174.8 53.2 41.4 26.0 21.6 23.6
31.3
174.1 54.4 28.0
174.7 52.5 41.6 26.1 22.0 23.7
175.1 54.0 41.8 26.1 22.2 23.8
δC
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)
3
2.32 2.46 3.98 1.48 1.47 1.35 1.30 1.17 1.52 1.30 1.30 1.13
4.27 1.65 1.65 0.94 0.94 8.27
dd (14.0, 8.5) dd (14.5, 4.5) m m m m brs m m brs brs m
dd (8.5, 6.0, α) m (β) m (γ) m (δ) m (δ) d (6.5)a
4.33 dd (9.0, 6.0, α) 1.95 m (β) 2.09 m (β) 2.41 m (γ) 8.23 d (6.5)a 176.7
4.42 1.65 1.65 0.94 0.94 8.12
4.45 1.65 1.65 0.94 0.94 7.97
δH
30.6−31.3 40.4 29.3 30.8 35.8 37.9
70.0 38.5 26.9
174.9 44.7
174.2 54.0 41.7 26.1 22.2 23.8
31.5
174.8 54.6 28.1
174.8 53.2 41.6 26.1 22.0 23.6
176.2 52.4 41.4 26.0 21.6 23.5
δC
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)a
m (α) m (β) m (γ) m (δ) m (δ) d (6.5)a
4
2.43 2.31 3.96 1.46 1.45 1.33 1.28 1.28 1.15 1.49 1.28 1.28
4.26 1.63 1.63 0.92 0.92 8.23
dd (4.5, 14.5) dd (8.5, 14.0) m m m m brs brs m m brs brs
dd (8.5, 6.0, α) m (β) m (γ) m (δ) m (δ) d (6.5)a
4.30 dd (9.0, 6.0, α) 1.92 m (β) 2.06 m (β) 2.39 t (4.5, γ) 8.20 d (6.5)a 176.8
4.39 1.63 1.63 0.92 0.92 8.09
4.42 1.63 1.63 0.92 0.92 7.93
δH
δC
30.7−31.2 30.7−31.2 40.4 29.3 30.9 35.8
70.0 38.4 26.9
174.9 44.7
174.2 54.0 41.7 26.1 22.2 23.8
31.4
174.8 54.6 28.3
174.8 53.2 41.6 26.1 22.0 23.6
176.3 52.4 41.3 26.0 21.6 23.5
Journal of Agricultural and Food Chemistry Article
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h in sterile distilled water. The motility bioassay was carried out with the zoospores. A series of solutions for gegeopeptides were prepared in aqueous dimethyl sulfoxide (DMSO). The final concentration of DMSO in the zoospore suspension was less than 1% (v/v), a condition that does not affect the motility of zoospores. The bioassay for the motility inhibition and lysis of zoospores was investigated as described previously.25,26 In brief, to a suspension (360 μL) of zoospores (ca.105/mL), 40 μL of sample solution was added and stirred with a glass rod. A 1% amount of aqueous DMSO was used as a control. Under a light microscope (×10 magnification), the time-course changes of motility and lysis of zoospores were evaluated as described previously.27 The bioassay was repeated twice, and each treatment was replicated thrice. The mean value (%) was calculated (±SE, standard error) of the affected spores in each treatment. Minimum inhibitory concentration (MIC) values of individual compounds were converted to micromolar.28 Further Activity Study against Bacteria and Fungi. A standard “broth dilution assay”29 was employed to evaluate the antimicrobial activity of gageopeptides against pathogenic fungi (R. solani, Botrytis cinerea (B. cinerea), and Colletotrichum acutatum (C. acutatum)), Gram positive bacteria (B. subtilis and Staphylococcus aureus (S. aureus)) and Gram negative bacteria (Salmonella typhi (S. typhi) and Pseudomonas aeruginosa (P. aeruginosa)). Briefly, a series of solutions for gegeopeptides were prepared over the range of 0.5−256 μg/mL in 96-well plates. Type strains were cultured overnight, and the final concentration (1.5 × 108 cfu/mL) of each strain in the culture broth was adjusted by comparing the culture turbidity with the 0.5 McFarland Standard. To each solution of gageopeptides 1−4, 30 μL of culture broth was added and the final volume was adjusted to 200 μL using the respective fresh culture medium. The plates were incubated for 24 h at 37 °C for bacteria and 48 h at 30 °C for fungi.28 The minimum inhibitory concentration (MIC, the lowest concentration of an antimicrobial compound that inhibits the visible growth of a microorganism) was recorded and expressed in micromolar in Result and Discussion.28 Cytotoxicity by SRB Assay. Cancer cell growth inhibitory activity of gageopeptides 1−4 was carried out according to a sulforhodamine B (SRB) assay.30 The selected cancer cell lines (brest cancer, MDA-MB231; colon cancer, HCT-15; prostate cancer, PC-3; lung cancer, NCIH23; stomach cancer, NUGC-3; and renal cancer, ACHN) were added into 96-well plates. A series of solutions of gageopeptides with the concentrations of 6.25−50 μM were prepared and added to the plates. The plates were incubated for 48 h, and anchorage-dependent cells were fixed with 50% (w/v) trichloroacetic acid and stained for 60 min. The SRB solution (0.4% sulforhodamine B in 1% acetic acid) was used to wash out excess dye. The protein-bound dye was dissolved in 10 mM Tris base solution for optical density determination at 510 nm using a microplate reader. Using graphed prism software, the GI50 values of gageopeptides were calculated. Cytotoxicity Test by WST-1 Assay. Inhibition of cancer cell growth for compounds 1−4 was determined by WST-1 (4-[3−4iodophenyl]-2-(4-nitrophenyl)-2H-5-tetrazolio)-1,3-benzene disulfonate) assay.31,32 In brief, the human myeloid leukemia K-562 cells were maintained in RPMI 1640 medium in 5% fetal bovine serum, supplemented with humidified air containing 5% CO2 at 37 °C. Compounds 1−4 were added to the properly distributed cells (2 × 104 cells/mL) in 96-well plates. The plates were incubated for 5 days under the preceding condition. A 50 μL aliquot of WST-1 stock solution (1.0 mg/mL) was added to the suspensions, and the plates were further incubated for 4 h. After aspirating off the supernatant with a microplate washer, the formazen crystals were dissolved by adding 150 μL of DMSO in each well. Finally, the absorbance was measured using a microplate reader at 480 nm, and IC50 values were calculated. Cytotoxicity Test by MTT Assay. The MTT assay was carried out to determine cancer cell viability.33 In brief, the RAW 264.7 cells (mouse leukemic monocyte macrophage cell line, 5 × 104 cells/mL), were cultured in a 96-well plate at 37 °C, and treated with varying concentrations (6.25, 12.5, 25, and 50 μM) of gageopeptides 1−4 for 24 h. Cells treated only with medium were used as a negative control group. The supernatant of each well was removed, and 20 μL of MTT
11.9 19.8 174.9
37.9
Article
19.8 23.2
11.9
0.87 m 0.87 m 14.6 23.5
30.6
0.89, t (7.5) 0.91, m 14.6 23.5
33.2
0.87 t 0.87 m 13 14 15
H NMR data recorded in CD3OH. a1
1.29 brs 12
no. units
Table 1. continued
δH
1
δC
1.29 m
δH
2
δC
1.30 brs 0.87 m
δH
3
δC
1.11 1.28 0.87 0.87 0.87
m brs m m m
δH
4
δC
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solution (5 mg/mL in phosphate buffered saline) and 100 μL of medium were added into the plates. The plates were incubated for another 4 h, and the formazen crystals were dissolved in DMSO (100 μL). The absorbance at 540 nm was measured using a microplate reader. The relative cell viability (%) was measured as a percentage relative to the untreated control cells.
acid residues. An additional oxygenated proton resonating at δH 3.98 displaying TOCSY and HMBC correlations with the carbonyl carbon at δC 175.0 and an aliphatic chain confirmed the presence of a fatty acid unit (e) in 1. The 13C NMR spectrum of 1 contained eight methyl carbons; six of them were found to constitute three Leu residues, and therefore, the remaining two methyls should be present in the fatty acid unit. The carbon resonating at δC 14.6 was assigned as a terminal methyl, and the methyl signal resonating at δC 23.5 was found to form a side chain with the fatty acid. By the detailed analysis of TOCSY and HMBC data, the position of the methyl group (δC 23.5) was determined to be at C-11 in the fatty acid chain. Judging from its molecular weight, the fatty acid was primarily assigned to be a C14 fatty acid connected to the N-terminal amine of the peptide. The LC-MS (m/z 243.01 [M − H]−) analysis of the acid hydrolysate of 1 confirmed its chain length. Thus, the fatty acid was assigned as a 3-β-hydroxy-11methyltridecanoic acid.20,21 Finally, the structure of 1 was determined to be Leu1-Leu2-Glu-Leu3-3-β-hydroxy-11-methyltridecanoic acid by HMBC and ROESY experiments (Figure 1). The molecular formula of 2 was determined to be C39H72N4O9 according to the HRESIMS result. The 1H and 13 C NMR data displayed a close similarity between 1 and 2 (Table 1), indicating the lipopeptidic nature of 2. However, the presence of an additional oxygenated methyl peak at δH 3.66 and the molecular weight deference of 2 (C2H4 more) from 1 prompted us to conduct a detailed investigation of its structure. Therefore, a series of 1D and 2D NMR experiments were performed to clarify the structure of 2. These NMR data analyses revealed the similar peptidic structure of 2, consisting of Leu and Glu residues, but the methoxy proton at δH 3.66 did not show any correlations in both COSY and TOCSY spectra. However, HMBC correlations between this proton and a carboxylic carbon at δC 176.7 were found, indicating the presence of a OMeGlu unit in 2. Based on the HMBC and ROESY spectra analysis, the sequence of these amino acid residues in the peptide moiety was assigned as Leu1-Leu2-Leu3OMeGlu. Similar to 1, a 3-β-hydroxy fatty acid was also determined from the observation of correlations of the oxygenated methine proton at δH 3.97 with the methylene protons at δH 2.32/2.46 in the COSY and TOCSY spectra, as well as with a carbonyl carbon at δC 174.9 in the HMBC spectrum. Initially, the elemental composition and molecular formula of 2 suggested that the fatty acid had a molecular formula of C15H30O3. Additional LC-MS results (m/z at 257.08 [M − H]−) of the fatty acid obtained from the acid hydrolysis of 2 confirmed the molecular formula. Thus, the fatty acid was determined as a 3-β-hydroxy-12-methyltetradecanoic acid. Finally, the structure of lipotetrapeptide 2 was constructed as Leu1-Leu2-Leu3-OMeGlu-3-β-hydroxy-12-methyltetradecanoic acid, based on the extensive 2D NMR data analysis (Figure 1). The molecular formula of 3 was assigned to be C37H68N4O9 by means of the HRESIMS result, which was the same as that of 1. Initially, the 1H NMR data of 3 were found very similar to 1. The difference between compounds 1 and 3 was the presence of several carbon resonances in the upfield region in the 13C NMR data of 3. The detailed analysis of a complete set of 1D and 2D NMR data of 3 indicated the presence of the same amino acid residues with the same sequence as 1. Moreover, the primary investigation on the molecular weight of 3 and further LC-MS analysis of the hydrolysate of 3 revealed that the molecular formula of the fatty acid in 3 was the same as
■
RESULTS AND DISCUSSION The molecular formula of 1 was found to be C37H68N4O9 based on the HRESIMS data analysis. The 1H NMR data (recorded in both CD3OD and CD3OH) analysis of pure compound 1 revealed its lipopeptidic nature (Table 1), showing amide proton signals at δH 7.99−8.28, α-methine groups at δH 4.26− 4.44, a broad singlet at 1.29, and methyl signals at δH 0.88− 0.97. This initial prediction of the lipopeptidic nature of 1 was further confirmed by the observation of carbonyl/carboxylic carbons at δC 174.2−176.7 and a group of CH2 carbons at δC 30.6−30.9 in the 13C NMR spectrum of 1, which were attributable to amino acid residues and an aliphatic chain, respectively. Later, the detailed interpretation of NMR data revealed five partial structures a−e, corresponding to four amino acid residues and a lipid moiety that constitute 1 (Figure 2). The glutamic acid unit (substructure c) was elucidated from
Figure 2. Assignment of partial structures by COSY and TOCSY correlations and complete structure with key HMBC and ROESY correlations of 1.
the TOCSY correlations of the amide proton at δH 8.23 with an α-methine proton at δH 4.33 and the methylene signals at δH 1.95/2.09, and the α-methine proton also showed TOCSY correlations with the methylenes at δH 1.95/2.09 and 2.41. Furthermore, HMBC correlations were observed between the methylene signals at δH 1.95/2.09 and 2.41 and a carboxylic carbon resonating at δC 176.7, and between the protons at δH 1.95/2.09 and a carbonyl carbon at δC 174.8, confirming the glutamic acid residue in 1. In a similar way, the substructures a−e were assigned by the analysis of 1D and 2D NMR data (Figure 2). The sequence of these amino acid residues was established as Leu1-Leu2-Glu-Leu3 by the detailed interpretation of ROESY and HMBC correlations between α-methines, NH protons, and amide carbonyl carbons of respective amino E
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Figure 3. Time-course motility inhibitory and lytic activities of (A) compound 1 and (B) compound 2 against zoospores of the late blight pathogen P. capsici at 0.02 μM (investigations were performed in triplicate).
Table 2. Inhibitory and Lytic Activities of Compounds 1 and 2 against P. capsici motility inhibitory and zoosporicidal activity over control (% ± SE)a 15 min dose (μM)
1
2
0.02 0.04 0.07 0.09 0.02 0.04 0.06 0.09 0.10
inhibited motility 74 89 100 100 0 20 69 84 100
± ± ± ± ± ± ± ± ±
whi6 8 0 0 0 3 6 7 0
30 min lysed 49 57 99 100 0 0 0 0 0
± ± ± ± ± ± ± ± ±
5 4 1 0 0 0 0 0 0
45 min
60 min
inhibited motility
lysed
inhibited motility
lysed
inhibited motility
lysed
87 ± 8 100 ± 0 100 ± 0 nt 41 ± 5 70 ± 8 82 ± 9 98 ± 2 100 ± 0
67 ± 6 92 ± 5 100 ± 0 nt 0±0 0±0 14 ± 5 25 ± 9 34 ± 4
92 ± 5 100 ± 0 nt nt 55 ± 6 88 ± 8 91 ± 9 98 ± 2 100 ± 0
79 ± 9 100 ± 0 nt nt 0±0 10 ± 4 21 ± 6 33 ± 7 49 ± 3
98 ± 2 nt nt nt 78 ± 6 92 ± 10 98 ± 3 100 ± 0 100 ± 0
86 ± 7 nt nt nt 0±0 21 ± 5 26 ± 5 45 ± 7 65 ± 4
Data presented here are the average value ± SE of at least three replications in each dose of compounds. nt, not tested. Compounds 3 and 4 did not show any activity against zoospores of P. capsici under the conditions tested.
a
Figure 4. Minimum inhibitory concentrations (μM) of 1−4 against (A) Gram positive and Gram negative bacteria and (B) fungi.
that of 1. However, the presence of additional methine carbons at δC 37.9 and 40.4 and methyl carbons at δC 11.9 and 19.8 in the 13C NMR spectrum indicated that 3 had a different fatty acid from 1. By the detailed interpretation of HMBC and TOCSY correlations, these additional methyl carbons were found to be located at C-9 and C-11 positions, and the fatty acid was assigned as a 3-β-hydroxy-9,11-dimethyltridecanoic acid.20 Finally, the structure of 3 was determined as Leu1-Leu2Glu-Leu3-3-β-hydroxy-9,11-dimethyltridecanoic acid by the extensive interpretation of 2D NMR data (Figure 1). The molecular formula of 4 was deduced to be C38H70N4O9 based on the HRESIMS data, and the 1H and 13C NMR data indicated that 4 was an additional analogue of 3. But, it was found that compound 4 had a molecular weight 14 Da more than 3, indicating the presence of an additional methylene group that could be present either in the fatty acid chain or in the peptide part. The careful interpretation of the NMR data
revealed that compounds 3 and 4 had the same peptide moiety, suggesting the additional CH2 group should be located in the fatty acid unit. This fact was further confirmed by the LC-MS analysis of the hexane phase of the acid hydrolysate of 4. Thus, the molecular formula of the fatty acid in 4 was determined to be C15H30O3. The fatty acid unit was found to possess same side chain as 3 with an additional methylene group and determined as a 3-β-hydroxy-9,11-dimethyltetradecanoic acid by 2D data analysis.21 Thus, the final structure of 4 was assigned as Leu1-Leu2-Glu-Leu3-3-β-hydroxy-9,11-dimethyltetradecanoic acid by HMBC and ROESY correlations (Figure 1). The absolute stereochemistry of the amino acid residues in 1−4 was found to be L-form by Marfey’s method. In addition, the absolute stereochemistry at C-3 of the fatty acid in 1 was assigned to be R by Mosher’s MTPA method.34 The absolute configuration of 3-hydroxy fatty acids in 2−4 (obtained from F
dx.doi.org/10.1021/jf502436r | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry the hydrolysis of 2−4 followed by extraction with hexane) was also found to be R by the comparison of their optical rotation values {[α]27D −22 (c o.1, MeOH), [α]27D −45 (c o.1, MeOH), and [α]27D −24 (c o.1, MeOH), respectively} with the optical rotation value [α]27D −26 (c o.1, MeOH) of the 3-hydroxy fatty acid in 1, as well as literature reviews, as the (R)-3-hydroxy fatty acid has the negative [α]D value in MeOH.20,21,35,36 Compounds 1−4 were tested against both bacteria and fungi. These compounds were found to inhibit the growth of these microorganisms. In particular, compounds 1−4 displayed significant activity against R. solani and P. capsici. Compound 1 showed both inhibitory and lytic activity against P. capsici at a concentration of 0.02 μM, whereas compound 2 showed only inhibitory activity at the same concentration (Figure 3). However, compound 2 exhibited lytic activities against P. capsici when the concentration was increased (Table 2). It may be noteworthy that compound 2 had an OMeGlu amino acid residue in the peptide unit and an additional CH2 group in the fatty acid chain compared to 1. It was also revealed that compounds 1 and 2 exhibited stronger antifungal activity than 3 and 4 (Figure 4B). Compounds 1 and 2 were structurally different from compounds 3 and 4 in terms of having less side chains in the fatty acid units, suggesting that the side chain length and the additional methyl group in the aliphatic chain are important for antifungal activity. Usually, the fungal cell wall consists of three main components, chitin, glucans, and proteins. Antifungal agents display their activity by disrupting the integrity of this membrane or inhibiting sterol synthesis. Conversely, lipopeptides inhibit the synthesis of β-1,3-D-glucan in the cell wall. In fact, without β-1,3-D-glucan, the integrity of the fungal cell wall is compromised, leading to increased osmotic fragility and the eventual lysis of the fungal cell.20,37 Since compounds 1−4 were found to exhibit potent antimicrobial activities, we became curious to know their possible cytotoxic effects. Accordingly, compounds 1−4 were tested against a panel of six human cancer cell lines according to a sulforhodamine B (SRB) assay. Interestingly, these compounds did not exhibit any activity (GI50 > 25 μM) against these cancer cell lines. The noncyotoxic character of these compounds was further checked against the leukemia cell line using WST-1 and MTT assays. From these detailed bioactivity results, it may be concluded that these lipotetrapeptides would be useful materials to develop environmentally friendly nontoxic antifungal agents.
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ACKNOWLEDGMENTS
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REFERENCES
This research was supported in part by the Korea Institute of Ocean Science and Technology (Grant PE99273 to H.J.S.).
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ASSOCIATED CONTENT
S Supporting Information *
Figures showing 1H, 13C, and 2D NMR spectra of 1−4, HPLC comparison of amino acid residues in 1 with Marfey’s derivatives of standard L- and D-amino acids, LC-MS data for 3 and 4. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel.: +82-31-400-6172. Fax: +82-31400-6170. Notes
The authors declare no competing financial interest. G
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