Secondary metabolites from micromonospora sp. (g044) - Cao Duc Tuan

Journal of Science and Technology 55 (3) (2017) 251-257 SECONDARY METABOLITES FROM MICROMONOSPORA SP. (G044) Cao Duc Tuan1, 3, Truong Bich Ngan1, Doan Thi Mai Huong1, *, Vu Thi Quyen1, Le Thi Hong Minh1, Brian Murphy2, Chau Van Minh1, Pham Van Cuong1, * 1Institute of Marine Biochemistry-VAST, 18, Hoang Quoc Viet, Cau Giay, Ha Noi, Viet Nam 2University of Illinois at Chicago, 700 S Halsted, Chicago, Illinois, USA 3Hai Phong University of Medicine and Pharmacy, 72A, Nguyen Binh Khiem, Ngo Quyen, Hai Phong, Viet Nam *Email: doanhuong7@yahoo.com Received: 24 August 2016; Accepted for publication: 28 December 2016 ABSTRACT In the course of our screening program, the EtOAc extract of a Micromonospora sp. (strain G044) from sponge Tethya aurantium of the sea of Côtô - Thanh Lân exhibited antimicrobial activity against Enterococcus faecalis, Bacillus cereus and Candida albicans. In this paper, we reported the isolation and structural elucidation of six secondary metabolites Cyclo-(Pro-Trp) (1), Cyclo-(Pro-Met) (2), Cyclo-(Pro-Val) (4), N-acetyltryptamine (3), uridine (5), and 2- phenylacetic acid (6) from the cultures broth of Micromonospora sp. (strain G044). The structures of 1 – 6 were determined by analyses of MS and 2D NMR data. All compounds were evaluated for their antimicrobial activity against a panel of clinically significant microorganisms. Compound 1 inhibited Escherichia coli with a MIC value of 128 µg/ml. Keywords: Micromonospora sp., marine microorganisms, antimicrobial activity, Cyclo-(Pro- Trp), Cyclo-(Pro-Met), Cyclo-(Pro-Val). 1. INTRODUCTION Sponges have been the focus of many studies as they have proved to be a rich source of biologically active secondary metabolites. Since the discovery of spongothymidine and spongouridine in the early 50's [1], a large number of biologically active compounds were isolated from marine sponges and their associated microorganisms [2]. The chemical diversity of sponge-derived products is remarkable and their biological activity ranges from anti- inflammatory, antitumour, immuno- or neurosuppressive, antiviral, antimalarial, antibiotic to antifouling [3]. However, many of the bioactive compounds found in sponges are, in fact, produced by their associated microbial communities [4 - 5]. In the course of our screening program, the EtOAc extract of a Micromonospora sp. (strain G044) from sponge of the sea of Côtô -Thanh Lân exhibited an inhibition activity against Enterococcus faecalis, Bacillus cereus and Candida albicans. In this paper, we reported the isolation and structural elucidation of six Cao Duc Tuan et al. 252 secondary metabolites Cyclo-(Pro-Trp) (1), Cyclo-(Pro-Met) (2), Cyclo-(Pro-Val) (4), N- acetyltryptamine (3), uridine (5), and 2-phenylacetic acid (6) from the cultures broth of Micromonospora sp. (strain G044) (Figure 1). Compound 1 inhibited Escherichia coli with a MIC value of 128 µg/ml. Figure 1. Compounds (1-6) isolated from the culture broth of Micromonospora sp (G044 strain). 2. MATERIALS AND METHODS 2.1. General Experimental procedures ESIMS were recorded on an Agilent 1100 LC-MSD Trap spectrometer. NMR spectra were recorded on a Bruker 500.13 MHz spectrometer operating at 125.76 MHz for 13C NMR, and at 500.13 MHz for 1H NMR. 1H chemical shifts were referenced to CDCl3, DMSO-d6 and CD3OD at δ 7.27, 2.50 and 3.31 ppm, respectively, while the 13C chemical shifts were referenced to the central peak of at δ 77.1 (CDCl3), 39.5 (DMSO-d6), and 49.0 (CD3OD). For HMBC experiments the delay (1/2J) was 70 ms. TLC silica gel Merk 60 F254 was used as Thin-layer chromatography. Column chromatography (CC) was carried out using silica gel 40 - 63 µm or Sephadex LH-20. 2.2. Marine Materials The samples of sponge Tethya aurantium (Pallas, 1767) were collected in Côtô - Thanh Lân in April 2014, and were identified by Prof. Do Cong Thung. Voucher specimens were deposited at the Institute of Marine Biochemistry and Institute of Marine Environment and Resources of the Vietnam Academy of Science and Technology, Hanoi, Vietnam. 2.3. Bacteria isolation, fermentation and identification The sponge sample (1 g) was added to the 10 mL of sterile sea water in a conical flask. The flask was agitated for about one hour. The sponge was filtered and the filtrate was serially diluted to obtain 10-1 to 10-7 dilutions using the sterilized sea water. An aliquot of 100 µL of each dilution was spread on the media. Different media like starch, yeast extract, peptone agar (A1), glycerol asparagine agar (GA Agar), humic acid-B vitamin agar (HV Agar) and glucose yeast malt extract agar (GYM) were used for isolation of actinomycetes. The media containing 50 % of sterile sea water were supplemented with rifampicin (5 µg/mL) and nystatin (25 µg/mL) (Himedia Mumbai) to inhibit bacterial and fungal contamination, respectively. The petriplates were incubated upto 3 weeks at 28 °C. The isolated discrete colonies were observed and used for identification. The fermentation of Micromonospora sp (strain G044) was cultured in high- nutrient medium (30 g of instant ocean, 10 g of starch, 4 g of yeast, 2 g of peptone, 1 g of Secondary metabolites produced by marine bacterirum Micromonospora sp. (G044) 253 calcium carbonate, 40 mg of iron sulfate, and 100 mg of potassium bromate) for 7 days at 28°C while shaking at 200 rpm. Sequencing 16S rRNA method was used for identification of strain G044. The 16S rRNA gene sequencing was carried by DNA Analyzer (ABI PRISM 3100, Applied Bioscience). Gene sequences were handled by BioEdit v.2.7.5. and compared with bacterial 16S rRNA sequences in GeneBank database by NBCI Blast program. 2.4. Extraction and isolation The culture broth (30 L) of Micromonospora sp. (strain G044) was filtered, and then extracted with ethyl acetate (15 L x 5 times). The extract was concentrated in vacuo to give 2.0 g of ethyl acetate extract. The EtOAc residue was chromatographed on silica gel column, eluted with a solvent gradient of CH2Cl2/MeOH (0 to 80 % MeOH in CH2Cl2) to yield 18 fractions. Fraction 7 (0.16 g) was chromatographed on a silica gel CC, eluted with mixtures of CH2Cl2/acetone (0 to 50 % acetone in CH2Cl2), to give five subfractions. Subfraction 7.5 was subjected to CC on silica gel, eluted with the mixture of CH2Cl2/acetone (95/5) to obtain 6 (6 mg). Fraction 11 (0.13 g) was subjected to Sephadex LH-20 CC (MeOH) to afford five subfractions. Subfraction 11.3 was purified by CC on silica gel, eluted with mixtures of CH2Cl2/acetone (95/5) to afford 2 (5 mg). Subfraction 11.5 was purified by CC on silica gel, eluted with the mixture of CH2Cl2/EtOAc (6/4) to afford 3 (4 mg) and 1 (8 mg). Fraction 15 was subjected to Sephadex LH-20 CC (MeOH) to afford four subfractions, subfraction 15.2 was purified by CC on silica gel, eluted with mixtures of CH2Cl2/acetone (9/1) to afford 4 (6 mg). Fraction 18 was subjected to Sephadex LH-20 CC (MeOH) to afford seven subfractions. Subfraction 18.6 was purified by TLC preparative using mixtures of CH2Cl2/acetone/MeOH (9/1/0.1) to afford 5 (3 mg). Cyclo-(Pro-Tryp) (1): White solid; ESI-MS: m/z 284.1409 [M-H]+; 1H-NMR (500 MHz, CD3OD): δH (ppm) 1.00 (1H, m, Ha-5), 1.52 (1H, m, Ha-4), 1.72 (1H, m, Hb-4), 1.99 (1H, m, Hb- 5); 3.27 (1H, m, Ha-3), 3.33 (1H, m, H-10), 3.48 (2H, m, Hb-3), 4.03 (1H, m, H-6), 4.43 (1H, m, H-9), 7.03 (1H, t, J= 8.0 Hz, H-5’), 7.11 (1H, t, J= 8.0 Hz, H-6’), 7.12 (1H, s, H-2’), 7.35 (1H, d, J = 8.0 Hz, H-7’), 7.59 (1H, d, J = 8,0 Hz, H-4’); 13C-NMR (150 MHz, CD3OD): δC (ppm) 22.6 (C-4), 29.1 (C-10), 29.1 (C-5), 45.9 (C-3), 57.2 (C-9), 60.1 (C-6), 109.6 (C-3’), 112.3 (C-7’), 119.8 (C-4’), 120.0 (C-5’), 122.6 (C-6’), 125.6 (C-2’), 128.7 (C-3’a), 138.0 (C-7’a), 167.5 (C=O); 170.8 (C=O). Cyclo-(Pro-Met) (2): White solid; ESI-MS: m/z 229.10 [M+H]+; 1H-NMR (500 MHz, CDCl3): δH (ppm) 1.90 (1H, m, Ha-4), 2.10 (2H, m, CH2-5), 2.11 (1H, m, Hb-4), 2.12 (3H, s, SCH3), 2.37 (2H, m, CH2-10), 2.68 (2H, t, J = 6.5 Hz, CH2-11), 3.54 (1H, m, Ha-3), 3.61 (1H, m, Hb-3), 4.10 (1H, m, H-6), 4.42 (1H, m, H-9); 13C-NMR (125 MHz, CDCl3): δC (ppm) 15.3 (SCH3); 22.7 (C- 4), 28.2 (C-5), 28.9 (C-11), 30.3 (C-10), 45.5 (C-3), 54.6 (C-9), 59.0 (C-6), 165.4 (C=O), 170.3 (C=O). 2-(3-indolyl) ethylamine acetate (3): White solid; ESI-MS: m/z 203 [M+H]+; 1H-NMR (500 MHz, CDCl3): δH (ppm) 1.93 (3H, s, COCH3), 2.98 (2H, t, J = 6.5 Hz, CH2-1’), 3.60 (2H, q, J = 6.5 Hz, CH2-2’), 7.04 (1H, d, J = 2.0 Hz, H-2), 7.13 (1H, td, J = 1.0, 8.0 Hz, H-5), 7.21 (1H, td, J = 1.0, 8.0 Hz, H-6), 7.38 (1H, d, J = 8.0 Hz, H-7), 7.60 (1H, d, J = 8.0 Hz, H-4), 8.16 (1H, br s, NH); 13C-NMR (125 MHz, CDCl3): δC (ppm) 23.4 (COCH3), 25.3 (C-1’), 39.8 (C-2’), 111.3 (C7), 113.0 (C-3), 118.7 (C-4), 119.5 (C-5), 122.0 (C-6), 122.3 (C-2), 127.4 (C-3a), 136.4 (C-7a), 170.2 (C=O). Cyclo-(Pro-Val) (4): White solid; ESI-MS: m/z 197.1[M+H]+; 1H-NMR (500 MHz, CD3OD): δH (ppm) 0.95 (3H, d, J = 6.8 Hz, CH3-11), 1.11 (3H, d, J = 6.8 Hz, CH3-12), 1.94 - 2.05 (3H, m, Cao Duc Tuan et al. 254 CH2-4, Ha-5), 2.33 (1H, m, Hb-5), 2.50 (1H, m, H-10), 3.54 (2H, m, H-3), 4.05 (1H, m, H-6), 4.22 (1H, m, H-9); 13C-NMR (125 MHz, CD3OD): δC (ppm) 15.2 (C-11), 17.4 (C-12), 21.8 (C- 4), 28.1 (C-5), 28.5 (C-10), 44.8 (C-3), 58.6 (C-9), 60.1 (C-6), 166.2 (C=O), 171.2 (C=O). Uridine (5): White solid; ESI-MS (m/z): 245.1 [M+H]+. 1H-NMR (500MHz, CD3OD) δ(ppm) 3.75 (1H, dd, J = 3.0, 12.5 Hz, Ha-5), 3.86 (1H, dd, J = 2.5, 12.5 Hz, H- Hb-5); 4.03 (1H, m, H-4), 4.17 (1H, m, H-3), 4.20 (1H, m, H-2), 5.72 (1H, d, J = 8.0 Hz, H-5), 5.92 (1H, d, J = 4.5 Hz, H-1), 8.02 (1H, d, J = 8.0 Hz, H-6). 2-phenylacetic acid (6): White solid. ESI-MS: m/z 136 [M]+. 1H-NMR (500 MHz, CDCl3): δH (ppm) 3.69 (2H, s, CH2-7); 7.30 - 7.39 (5H, m, Ph-H). 13C-NMR (125 MHz, CDCl3): δC (ppm) 41.4 (C-7), 127.2 (C-4), 128.6 (C-2 and C-6), 129.4 (C-3 and C-5), 133.8 (C-1), 175.2 (COOH). 2.5. Antimicrobial assay Antimicrobial assays were carried out using E. coli (ATCC25922), P. aeruginosa (ATCC27853), S. enterica (ATCC12228), E. faecalis (ATCC13124), S. aureus (ATCC25923), B. cereus (ATCC13245), and C. albicans (ATCC1023). Stock solutions of samples were prepared in DMSO, and the antimicrobial assays were carried out in 96-well microtiter plates against the microbial strains (5 × 105 CFU/mL) using a modification of the published method [6]. After incubation for 24 h at 37 °C, the absorbance at 650 nm was measured using a microplate reader. Streptomycin and nystatin were used as reference compounds. 3. RESULTS AND DISCUSSION Compound 1 was isolated as a white solid. In its positive HRESI mass spectrum, the pseudo- molecular ion was observed at m/z 284.1409 [M+H]+, suggesting a molecular formula of C16H19N3O2. Analyses of the 13C-NMR and DEPT spectra with the aid of the HSQC of 1 indicated the presence of 16 carbons, including four sp3 methylenes at δC 22.6 (C-4), 29.1 (C- 10), 29.1 (C-5), 45.9 (C-3), two sp3 methines at δC 57.2 (C-9), 60.1 (C-6), eight aromatic carbons (five methines and three quaternary carbons) and two carbonyl carbons at δC 167.4 and 170.3. The 1H-NMR spectrum of 1 indicated the presence of a 1,2-disubstituted benzene ring [δH 7.03 (1H, t, J= 8.0 Hz, H-5’), 7.11 (1H, t, J= 8.0 Hz, H-6’), 7.35 (1H, d, J= 8.0 Hz, H-7’), 7.59 (1H, d, J= 8,0 Hz, H-4’)], a singlet aromatic proton at δH 7.12 (1H, s, H-2’), and ten protons in the aliphatic region. The chemical shifts of CH-6 (δC 60.7, δH 4.03), CH-9 (δC 57.2, δH 4.42), and CH2-3 (δC 45.9, δH 3.27, 3.48) suggested their linkage to nitrogen. Analysis of COSY spectrum revealed the presence of three spin – spin coupling systems: CH2-3/CH2-4/CH2-5/CH-6; CH- 9/CH2-10 and H-4’/H-5’/H-6’/H-7’ (Figure 2). These observed data revealed that compound 1 was a cyclodipeptide compound, forming from a proline and tryptophan units. In the HMBC spectrum, the correlation of H-10 with C-2, C-3’, C-9, C3a’and C-1 suggested that the indole ring was attached to C-10 (Figure 2). Detailed analysis of 2D NMR spectra, especially HMBC spectrum allowed determining the structure of 1 as Cyclo-(Pro-Trp). This cyclodipeptide was previously described [5, 7]. Secondary metabolites produced by marine bacterirum Micromonospora sp. (G044) 255 Figure 2. Selected COSY ( ) and HMBC ( ) correlations of 1. Compound 2 was obtained as a white amorphous solid. The ESI-MS indicated the pseudomolecular ion peak at m/z 229.10 [M+H]+. The 1H-NMR spectrum of 2 displayed signals of a methyl groups at δH 2.12 (3H, s, SCH3) and signals of 12 aliphatic protons ranging from 1.90 to 4.42 ppm. Analysis of the 13C NMR and DEPT spectra of 2 revealed the presence of 10 carbons, including one methyl groups at δC 15.3 (SCH3), two methines at δC 54.6 (C-9), 59.0 (C- 6), five methylenes at δC 22.7 (C-4), 28.2 (C-5), 28.9 (C-11), 30.3 (C-10), 45.5 (C-3), and two carbonyl at δC 165.4 (C=O) and 170.3 (C=O). The chemical shifts of CH-6 (δC 59.0, δH 4.10), CH-9 (δC 54.6, δH 4.42) and CH2-3 (δC 45.5, δH 3.54 and 3.61) suggested their linkage to nitrogen atoms. Detailed analysis of the NMR suggested the structure of 2 which was a cyclodipeptide compound, forming from a proline and methionine units. Complete analysis of the NMR spectra and comparison with reported NMR data indicated that compound 2 was Cyclo-(Pro-Met) [8]. Compound 3 was isolated as a white solid. In its positive ESI mass spectrum, the pseudo- molecular ion was observed at m/z 203 [M+H]+. In the 1H-NMR spectrum, the presence of a 1,2- disubstituted benzene ring [δH 7.13 (1H, td, J = 1.0, 8.0 Hz, H-5), 7.21 (1H, td, J= 1.0, 8.0 Hz, H-6), 7.38 (1H, d, J = 8.0 Hz, H-7), 7.60 (1H, d, J = 8.0 Hz, H-4)], and a singlet aromatic proton at δH 7.04 (1H, d, J = 2.0 Hz, H-2) were noted. However, at the aliphatic region of in the 1H- NMR of 3, the signals of an acetyl at δH 1.93, a triplet methylene group at δH 2.98 and a quartet methylene group bonded with nitrogen at δH 3.60 were observed. The 13C-NMR and DEPT spectra of 3 showed signals of the groups observed above with additional signals of a caboxyl amide group at δC 170.2 and three quaternany carbone. Complete analysis of the NMR spectra and comparison with reported NMR data indicated that compound 3 was 2-(3-indolyl) ethylamine acetate [9]. Compound 4 was isolated as a white solid. The ESI mass spectrum of 1 presented a base peak at m/z 197.1 [M+H]+. In the 13C-NMR spectrum, the some signals of protons at the aliphatic region of 4 was close of 1, including one methylene group attached to nitrogen at δC 44.8 (C-3), 2 methine sp3 groups bonded with nitrogen at δC 58.6 (C-9) and δC 60.1 (C- 6), two carbonyl amide groups at δC 166.2 (C=O); 171.2 (C=O) and two methylen groups at δC 21.8 (C- 4), 28.1 (C-5). The difference between two compounds was the presence of a -CH(CH3)2 moiety of 4 instead of a indole ring of compound 1, this observation was based on 1H-NMR spectra, the signals of two doublet methyl groups at 0.95 (3H, d, J=6.8 Hz, CH3-11), 1.11 (3H, d, J = 6.8 Hz, CH3-12) and a methine group at 2.50 (1H, m, H-10) instead of the indol ring signals of 1. These results suggested 4 belonging to a diketopiperazine compound. NMR-data comparison with those previously reported defined compound 4 to be identical with Cyclo-(Pro-Val) [10-11]. Cao Duc Tuan et al. 256 Compound 5 was obtained as a white solid. The ESI mass spectrum of 5 showed a base peak at m/z 245.1 [M+H]+. In the 1H-NMR, compound 5 displayed signals of two olefin protons at δH 5.72 (1H, d, J= 8.0 Hz, H-5), 8.02 (1H, d, J= 8.0 Hz, H-6) and a set of the protons of arabionoside sugar moiety at δH 3.75 (1H, dd, J=3.0, 12.5 Hz, Ha-5), 3.86 (1H, dd, J = 2.5, 12.5 Hz, Hb-5); 4.03 (1H, m, H-4); 4.17 (1H, m, H-3 ); 4.20 (1H, m, H-2); 5.92 (1H, d, J = 4.5 Hz, H-1). Comparison of the 1H-NMR spectrum and TLC of 5 with uridine which was available in our laboratory revealed their similarity. Thus, 5 was determined as uridine [12]. Compound 6 was isolated as a white solid. The ESI mass spectrum of 6 presented a base peak at m/z 138 [M+H]+. In the 1H-NMR, compound 6 displayed signals of 5 aromatic protons at δH 7.30-7.39 (5H, m, Ph-H), one singlet methylene at δH 3.59 (CH2-7’). The 13C-NMR and DEPT spectra of 2 indicated the presence of a phenyl ring at δC 127.2-133.8, one methylene group at δC 41.4 (C-7) and a carbonyl group at δC 175.2. Complete analysis of NMR spectra and comparison with the data reported in the literature allowed determining the structure of 6 to be 2-phenylacetic acid [13]. All the isolates were evaluated for their antibacterial activity against Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Salmonella enterica (ATCC12228), Enterococcus faecalis (ATCC13124), Staphylococcus aureus (ATCC25923), Bacillus cereus (ATCC13245), and antifungal activity against Candida albicans (ATCC1023). Compounds 1 selectively inhibited E. coli with MIC value of 128 µg/mL, in comparison with the reference compound, streptomycin (MIC: 32 µg/mL). 4. 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