Secondary metabolites produced by marine actinomycetestreptomycessp G246

Vietnam Journal of Science and Technology 59 (1) (2021) 1-8 doi:10.15625/2525-2518/59/1/15176 SECONDARY METABOLITES PRODUCED BY MARINE ACTINOMYCETESTREPTOMYCESSP. G246 Do Thi Quynh 1, 2 , Doan Thi Mai Huong 1, 2, * , Tran Van Hieu 1 , Truong Bich Ngan 1 , Le Thi Hong Minh 1 , Vu Thi Quyen 1 , Nguyen Thi Hoang Anh 3 , Brian T. Murphy 4 , Pham Van Cuong 1, 2, * 1 Institute of Marine Biochemistry, Vietnam Academy of Science and Technology (VAST), 18 Hoang QuocViet, Cau Giay, Hanoi 10000, Viet Nam 2 Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam 3 Thaibinh Medical College, 290 Phan Ba Vanh, Quang Trung, Thai Binh, Viet Nam 4 Department of Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60607, USA * Email: huongdm@imbc.vast.vn Received: 25 June 2020; Accepted for publication: 1 September 2020 Abstract. In a recent study, we described two new lavandulylated flavonoids, along with eight known compounds from the culture broth of a Streptomyces sp. (strain G246), isolated from the sponge Halichondria panicea, collected in the sea of Son Tra peninsula (Da Nang). A comparison study was conducted to differentiate between solid and liquid fermentation technique for secondary metabolites production of strain G246. In this paper, we report the isolation and structural characterization of nine secondary metabolites (1-9) from strain G246 by solid state fermentation. Compound 2 was the only one similarity between these fermentation techniques. Keywords: Streptomyces, marine microorganism, actinomycete, solid state fermentation, secondary metabolites. Classification numbers: 1.1.1, 1.5.3. 1. INTRODUCTION Fermentation of micro-organisms has been widely used for the production of antibiotics and other bioactive compounds. The development of techniques such as solid state fermentation and liquid fermentation led to industrial-level for production of biological active compounds [1, 2]. At laboratory scale, in general, liquid fermentation is used more often for researches on secondary metabolite productions [3, 4]. Recently, we described two new lavandulylated flavonoids, along with eight known compounds from the culture broth of a Streptomyces sp. (strain G246) isolated from sponge Halichondria panicea, collected in the sea of Son Tra peninsula (Da Nang) [4]. In this paper, we report the isolation and structural characterization of 9 compounds including spirotry prostatins A (1), cyclo-(Pro-Met) (2), phenol A acid (3), 3,4- Doan Thi Mai Huong, et al. 58 dihydroxy-6,7-dimethyl-quinoline-2-carboxylic (4), cyclo-(Pro-Gly) (5), xanthone (6), guanosine (7), 2′-deoxyadenosine (8), adenosine (9) from Streptomyces sp. (strain G246) by solid state fermentation technique (Figure 1). The study aim was to delineate the differences between solid state and liquid fermentation for secondary metabolites production of the strain G246. Indeed, there was only one similarity, compound (2), found from these fermentation techniques. Figure 1. Nine compounds (1-9) from marine Streptomyces sp. G246 isolated by solid state fermentation technique. 2. MATERIAL AND METHODS 2.1. General experimental procedures The ESI-MS 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 13 C NMR, and at 500.13 MHz for 1 H-NMR. The 1 H chemical shifts were referenced to CDCl3and CD3OD at δH 7.27 and 3.31 ppm, respectively, while the 13 C chemical shifts were referenced to the central peak of at δC 77.1 (CDCl3) and 49.0 (CD3OD). For HMBC experiments the delay (1/2J) was 70 ms. TLC silica gel Merck 60 F254 was used as thin layer chromatography. Column chromatography (CC) was carried out using silica gel 40 - 63 µm, YMC RP-18 (30-50 µm) or Sephadex LH-20. 2.2. Marine materials The strain G246 was isolated from a sample of sponge Halichondria panicea collected in the sea of Son Tra island (Da Nang, Viet Nam), August 2016. Marine sample was identified by Prof. Do Cong Thung of Institute of Marine Environment and Resources - Vietnam Academy of Science and Technology (VAST). Voucher specimens were deposited at the Institute of Marine Environment and Resources, Hai Phong, Viet Nam. Strain G246 was later identified belonging to Streptomyces genus based on its 16S rRNA gene sequence (GeneBank Access Code: MG917689) [4]. Secondary metabolites produced by marine actinomycete Streptomycessp. G246 59 2.3. Solid state fermentation Strain G246 was firstly activated and inoculated into 1 L of A1 medium, pH 7.0, comprising: soluble starch: 5 g, yeast extract: 2 g, peptone:1 g and instant ocean: 30 g in 1 L of distilled water. After 7 days of incubation at 28 o C with agitation of 200 rpm, the culture broth was spread on 50 flasks containing 1L of high-nutrient solid medium A1+ (soluble starch: 10 g/L, yeast extract: 4 g/L, peptone: 2 g/L, instant ocean: 30 g/L, CaCO3: 1 g/L, agar: 15 g/L, and water: 1 L). The fermentation was incubated in an incubator at 28 o C and harvested on the tenth day. 2.4. Extraction and isolation The culture agar of Streptomycessp. G246 strain was minced, and extracted successively with ethyl acetate and methanol (three times × 30 minute each) at 40 o C over sonication, and concentrated under reduced pressure to obtain corresponding extracts: ethyl acetate (EG246, 17.2 g) and methanol (MG246, 20.2 g). There were no differences between EG246 and MG246after checking with TLC, so they were combined to obtain a single extract (S246). The S246 was separated by a reversed phase (RP) C18 column chromatography (CC), eluting with MeOH/H2O gradient to provide eleven fractions (F1- F11). Fraction F4 (1.1 g) was separated into four sub-fractions (F4.1–F4.4) by Sephadex LH-20 column using MeOH as eluent. Purification of the sub-fraction F4.4 (100 mg) by Sephadex LH-20 column using MeOH to give compound 7 (10 mg). Separation of fraction F10 (0.61 g) by Sephadex LH-20 column using MeOH to obtain six sub-fractions (F10.1-F10.6). The sub-fraction F10.4 (100 mg) was subjected to Sephadex LH-20 column using MeOH as mobile phase to give compound 8 (15 mg). Sub- fraction F10.5 (200 mg) was separated on Sephadex LH-20 column, eluting by MeOH to give compound 9 (11 mg). Fraction F11 (6.2 g) was submitted to a silica gel CC, eluted with CH2Cl2/MeOH gradient to yield four sub-fractions (F11.1-F11.4). Sub-fraction F11.2(1.2 g) was further purified by silica gel CC, eluted with CH2Cl2/EtOAc gradient to give five sub-fractions (F11.2.1-F11.2.5). Sub-fraction F11.2.3 (400 mg) was further separated on Sephadex LH-20 column using MeOH as eluent to give compound 5 (17 mg). Sub-fraction F11.2.4 (200 mg) was purified on silica gel CC, eluted with CH2Cl2/EtOAc gradient to give compound 4 (2.7 mg). Sub-fraction F11.2.5 (100 mg) was subjected to CC on silica gel, eluted with CH2Cl2/acetone gradient to provide compound 1 (8.5 mg) and compound 6 (5 mg). Sub-fraction F11.3 (2.0 g) was chromatographed on silica gel column, eluting with CH2Cl2/MeOH gradient (0-50% MeOH) to give five sub-fractions (F11.3.1.1-F11.3.5). Purification of the sub-fraction F11.3.4 (500 mg) by silica gel CC, eluted with CH2Cl2/acetone gradient to give compound 2 (7.0 mg). Sub- fraction F11.3.5 (200 mg) was further separated on silica gel CC, eluted with CH2Cl2/MeOH gradient to give compound 3 (4.7 mg). Spirotryprostatin A (1): White solid; [α]29D -29.5 (c 0.1 MeOH). ESI-MS (m/z): 396 [M+H] + . 1 H-NMR (500 MHz, CD3OD): δH 1.20 (3H,s, H-21); 1.67 (3H, s, H-22), 2.03 (1H, m, Ha-14), 2.14 (1H, m, Hb-14), 2.15 (1H, m, Ha-13), 2.32 (1H, m, Hb-13), 2.37 (1H, dd, J = 7.0,13.5 Hz, Ha-8), 2.61 (1H, dd, J = 10.5,13.5 Hz, Hb-8), 3.57 (2H, m, H-15), 3.80 (OCH3), 4.45 (1H, t, J = 8.0 Hz, H-12), 4.73 (1H, d, J = 9.0 Hz, H-18), 5.04 (1H, dd, J = 10.0,7.5 Hz, H-9), 5.09 (1H, d, J = 9.0 Hz, H-19), 6.51 (1H, d, J= 2.0 Hz, H-7), 6.56 (1H, dd, J = 2.0,7.5 Hz, H-5), 7.03 (1H, d, J = 7.5 Hz, H-4). 13 C-NMR (125 MHz, CD3OD): δC 18.09 (C-22), 24.44 (C-14), 25.54 (C-21), 28.52 (C-13), 35.33 (C-8), 46.18 (C-15), 55.93 (OCH3), 57.11 (C-3), 59.81 (C- 9),61.83 (C-18), 62.26 (C-12), 97.78 (C-7), 107.77 (C-5), 120.13 (C-3a), 122.67 (C-19), 128.20 (C-4), 139.10 (C-20), 144.35 (C-7a), 162.16 (C-6), 168.94 (C-17), 169.45 (C-11), 183.20 (C-2). Doan Thi Mai Huong, et al. 60 Cyclo-(Pro-Met) (2): White solid, ESI-MS (m/z): 229 [M+H] + . 1 H-NMR (500 MHz, CDCl3): δH (ppm) 1.90 (1H, m, Ha-4), 2.01 (2H, m, Hb-4,Ha-5), 2.11 (1H, m, Hb-5),2,12 (3H, s, SCH3), 2.37 (2H, m, CH2-10), 2.68 (2H, t, J = 7.0 Hz, CH2-11), 3.54 (1H, m, Ha-3), 3.60 (1H, m, Hb-3), 4.10 (1H, t, J = 8.0 Hz,, H-6), 4.20 (1H, m, H-9), 6.77 (1H, br. s, NH). 13 C-NMR (125 MHz, CDCl3): δC (ppm) 15.30 (SCH3), 22.67 (C-4), 28.21 (C-5), 28.88 (C-11), 30.27 (C-10), 45.49 (C-3), 54.65 (C-9), 59.03 (C-6), 165.40 (C=O), 170.33 (C=O). Phenol A acid (3): White solid, [α]29D -45.5 (c 0.1 MeOH). HR-ESI-MS (m/z): 503.1881 [2M+Na] + (Calcd. for C24H32O10Na, m/z 503.1893). 1 H-NMR (500 MHz, CD3OD): δH1.59 (3H, d, J = 6.5 Hz, Me-9), 1.67 (3H, d, J = 6.5 Hz, Me-10),2.11 (3H, s, Me-11), 3.10 (1H, q, J = 7.0 Hz, H-7), 3.91 (1H, q, J = 7.0 Hz, H-8), 6.25 (1H, s, H-5). 13 C-NMR (125 MHz, CD3OD): δC10.60 (C-11), 16.37 (C-10), 19.81 (C-9), 43.41 (C-7), 71.87 (C-8), 103.51 (C-1), 104.69 (C-5), 114.15 (C-3), 149.72 (C-4), 160.32 (C-6), 160.80 (C-2), 173.04 (COOH). 3,4-dihydroxy-6,7-dimethyl-quinoline-2-carboxylic (4): Yellow solid, mp. 153 – 154 oC. ESI-MS (m/z): 234 [M+H] + . 1 H NMR (500 MHz, DMSO-d6): δH2.46 (3H, s, Me-10), 2.48 (3H, s, Me-11), 7.69 (1H, s, H-8), 7.89 (1H, s, H-5), 11.54 (1H, br, s, OH). 13 C NMR (125 MHz, DMSO-d6): δC 19.43 (C-10), 20.08 (C-11), 125.89 (C-8), 128.70 (C-5),129.83 (C-4a), 138.47 (C- 8a), 139.10 (C-6), 141.69 (C-4), 144.92 (C-7), 146.28 (C-2), 149.89 (C-3), 160.52 (C-9). Cyclo-(Pro-Gly) (5): White solid, mp. 210 – 211 oC, [α]D 25 -142,5 o (c 0.40, MeOH). ESI- MS (m/z): 155 [M+H] + . 1 H-NMR (500 MHz, CDCl3): δH 2.01 (3H, m, CH2-4, H-5a),2.34 (1H, m, H-5b), 3.56 (2H, m, CH2-3), 3.77 (1H, d, J = 17.0 Hz,H-9a), 4.13 (1H, d, J = 17.0 Hz,H- 9b), 4.25 (1H, td, J = 2.0,8.0 Hz, H-6), 13 C-NMR (125 MHz, CDCl3): δC 23.28 (C-4), 29.36 (C- 5), 46.30 (C-3),47.00 (C-9), 59.84 (C-6), 166.44 (C-1), 171.98 (C-7). Xanthone (6): ESI-MS (m/z): 195 [M-H] - . 1 H-NMR (500 MHz, CDCl3): δH (ppm) 6.67 (2H, m, H-2, H-4), 7.30 (1H, t, J = 7.5 Hz, H-3), 7.92 (1H, d, J = 7.5 Hz, H-1). 13 C-NMR (125 MHz, CDCl3): δC (ppm) 109.78 (C-9a), 116.49 (C-2), 116.80 (C-4), 132.14 (C-1), 135.00 (C- 3),151.06 (C-4a),173.0 (C-9). Guanosine (7): White solid. ESI-MS (m/z): 284 [M+H] + . 1 H-NMR (500 MHz, DMSO-d6): δH 3.52 (1H, dt, J = 4.0, 12.0 Hz, Ha-5′), 3.62 (1H, dt, J = 4.5, 12.0 Hz, Hb-5′), 3.87 (1H, dt, J = 4.0, 3.5 Hz, H-4′), 4.08 (1H, br s, H-3′), 4.39 (1H, m, H-2′), 5.69 (1H, d, J = 6.0 Hz, H-1′), 7.92 (1H, s, H-8). 13C-NMR (125 MHz, DMSO-d6): δC 61.39 (C-5′), 70.35 (C-3′), 73.68 (C-2′), 85.18 (C-4′), 86.35 (C-1′), 116.68 (C-5), 135.54 (C-8), 151.28 (C-4), 153.65 (C-2), 156.73 (C-6). 2′-Deoxyadenosine (8): White solid, ESI-MS (m/z): 252 [M+H]+. 1H-NMR (500 MHz, CD3OD): δH 2.43 (1H, m, Ha-2′), 2.84 (1H, m, Hb-2′), 3.76 (1H, dd, J = 3.5, 12.5 Hz, Ha-5′), 3.87 (1H, dd, J = 3.0, 12.5 Hz, H- Hb-5′), 4.09 (1H, m, H-3′), 4.60 (1H, m, H-4′), 6.45 (1H, dd, J=6.0, 8.0 Hz, H-1′),8.20 (1H, s, H-8), 8.34 (1H, m, H-2).13C-NMR (125 MHz, CD3OD): δC (ppm) 41.64(C-2′), 63.58 (C-5′),73.12 (C-3′), 87.11 (C-4′), 89.91 (C-1′),120.81 (C-5),141.53 (C-8), 149.87 (C-4), 153.49 (C-2), 157.53 (C-6). Adenosine (9): White solid, ESI-MS (m/z): 268 [M+H] + , 1 H-NMR (500 MHz, CD3OD):δH3.77 (1H, dd, J = 3.0, 12.5 Hz, Ha-5′), 3.91 (1H, dd, J = 2.5, 12.5 Hz, Hb-5′), 4.19 (1H, m, H-4′), 4.35 (1H, dd, J = 3.0, 5.0 Hz, H-3′), 4.76 (1H, dd, J = 5.0, 6.5 Hz, H-2′ ), 5.99 (1H, d, J = 6.0 Hz, H-1′), 8.20 (1H, s, H-8), 8.32 (1H, s, H-2). 3. RESULTS AND DISCUSSION Compound 1 was obtained as white solid with an [α]29D value of -29.5 (c 0.1 MeOH). The ESI-MS spectra of 1 showed the pseudomolecular ion peak at m/z 396 [M+H] + . Analysis of the Secondary metabolites produced by marine actinomycete Streptomycessp. G246 61 13 C NMR and DEPT spectrum of 1 revealed the presence of 22 carbons, including three carbonyl carbons at δC 168.94 (C-17), 169.45 (C-11), 183.20 (C-2), four sp 2 methines at δC 97.78(C-7), 107.77 (C-5), 122.67 (C-19), 128.20 (C-4), three sp 3 methines at δC59.81 (C-9), 61.83 (C-18), 62.26 (C-12), four sp 3 methylenes at δC 24.44 (C-14), 28.52 (C-13), 35.33 (C-8), 46.18 (C-15), one methoxy group at δC 55.93, two methyls at δC 18.09 (C-22), 25.54 (C-21) and five quaternary carbons. The 1 H-NMR spectrum of 1 indicated the presence of a 1,2,4-trisubstituted benzene ring at δH 6.51 (1H, d, J = 2.0 Hz. H-7), 6.56 (1H, dd, J = 2.0 and 7.5 Hz, H-5), 7.03 (1H, d, J = 7.5 Hz, H-4), one olefinic proton at δH 5.09 (1H, d, J = 9.0 Hz, H-19), one methoxy group at δH 3,80 (3H, s, OCH3), two singlet methyls at δH 1.20 (3H,s, H-21), 1.67 (3H, s, H-22) and a set of protons at the aliphatic region. In the 1 H- 1 H COSY spectrum, in addition to the correlations of protons of ABX aromatic ring, three separated spin-spin coupling systems was observed as follows: i) H-12 (δH 4.45)/H-13 (δH 2.15 and 2.32)/H-14 (δH 2.14 and 2.03)/H-15 (δH 3.57), ii) H-8 (δH 2.61 and 2.37)/H-9 (δH5.04), iii) H-18 (δH4.73)/H-19 (δH 5.09) (Figure 2). Analysis of the HMBC spectrum of 1 noted cross-peak of H-12 (δH 4.45) and H-13 (δH 2.15 and 2.32) with C-11 (δC169.45) and H-15 (δH 3.57) with C-17 (δC168.94). This observation demonstrated the presence of proline moiety of a cyclodipeptide molecule. Similarly, HMBC correlations of proton of methoxy group, H-4, H-5 and H-7 with C-6 (δC162.16) indicated the linkage of methoxy group at C-6 of ABX aromatic ring. HMBC correlations of H-21 (δH 1.20) and H-22 (δH 1.67) with C-19 (δC122.67), C-20 (δC 139.10) assigned the side chain as figure (Figure 2). Finally, cross-peak of H-8 with C-2 (δC183.20), C-3 (δC57.11), C-3a (δC120.13), C- 17 (δC168.94), C-18 (δC61.83), cross-peak of H-18 (δH 4.73) with C-2 (δC 183,2), C-3 (δC 57,1),C-8 (δC 35,3), C-9 (δC 59,8), C-11 (δC169.45), cross-peak of H-9 with C-17 (δC168.94), C- 11 (δC 169.45) established the second moiety of the cyclodipeptide molecule with spiro ring system as figure (Figure 2). On the basis of the above NMR, MS spectra, value of [α]29D and comparison with reported literature [5], compound 1 was determined asspirotryprostatin A. Figure 2. Key COSY and HMBC correlations for compounds of 1, 3 and 7. Compound 2 was isolated as a white solid. Its positive ESI mass spectrum showed the proton adduct ion [M+H] + at m/z 229 [M+H] + . The 13 C NMR and DEPT spectra of 2 showed the presence of 10 carbons, includingtwo carbonyl carbons at δC 165.40 (C=O) and 170.33 (C=O), two sp 3 methines bearing nitrogen at δC 54.65 (C-9), 59.03 (C-6), five sp 3 methylenes and one singlet methyl at δC15.30 (SCH3). The 1 H-NMR spectrum of 2 showed the presence of one amide NH at 6.77 (1H, br s, NH), one singlet methyl at δH 2,12 (3H, s, SCH3), one triplet methylene at δH 2.68 (2H, t, J=7.0 Hz, H-11), two sp 3 methines at δH 4.10 (1H, t, J = 8.0 Hz,, H- 6), 4.20 (1H, m, H-9) and the 8 other protons at the aliphatic region. This observation suggested compound 2 is a cyclodipeptide with the presence of two amino acid moieties: proline and Doan Thi Mai Huong, et al. 62 methionine. This analysis was further supported by comparison with reported data [6]. Thus, compound 2 was defined as cyclo-(Pro-Met). Compound 3 was isolated as a white solidand was optically active [α]29D -45.5 (c 0.1 MeOH). The molecular formula of 3 was determined to be C12H16O5 on the basis of the pseudo- molecular ion [2M+Na] + at m/z503.1881(calcd. for C24H32O10Na, m/z 503,1893) in HR-ESI-MS spectrum and 13 C-NMR datas. The 1 H-NMR spectrum of 3 revealed the signals of one singlet proton aromatic at δH 6.25, suggesting the presence of a 1,2,3,4,6-pentasubstituted benzene ring, one singlet methyl at δH2,11(3H, s, Me-11), two doublet methyls at δH 1.59 (3H, d, J = 6.5 Hz, Me-9), 1.67 (3H, d, J = 6.5 Hz, Me-10) and signals of two sp 3 methines at δH 3.10 (1H, q, J = 7.0 Hz, H-7), 3.91 (1H, q, J = 7.0 Hz, H-8). The 13 C-NMR and HSQC spectra of 3 showed the presence of 12 carbons including ones p 2 methine group at δC104.69 (C-5), one carboxyl group at δC 173.04 (COOH), three methyl groups at δC 10.60 (C-11), 16.37 (C-10), 19.81 (C-9), two sp 3 methine which one was linked to the oxygen at δC43.41 (C-7), 71.87 (C-8), and five quaternary carbons. In the HMBC spectrum of 3, cross-peaks ofthe proton of CH3-11 (δH2.11) with C-2 (δC 160.80), C-3 (δC 114.15), C-4 (δC 149.72) indicated the linkage of this methyl group at C-3. Futhermore, HMBC corelation of the proton of CH3-10 (δH 1.67) with C-7(δC 43.41)/C-4(δC 149.72), C-8 (δC 71.87) indicated the linkage of this methyl group at C-7. The cross peak of H-7 (δH 3.10) with C-4 (δC 149.72), C-3 (δC 114.2), C-5 (δC 104.69) determined the linkage of the C-7 with C-4 of aromatic ring (Figure 2). Thus, complete analyses of the 2D- NMR spectra, and comparison with the reported data allowed determining the structure of 3 to be phenol A acid [7]. Compound 4 was isolated as yellow solid. Its ESI mass spectrum showed the pseudo- molecular ion [M+H] + at m/z 234. The 1 H NMR spectrum of 4 showed signals of two singlet aromatic protons at δH7.89 (1H, s, H-5) and 7.69 (1H, s, H-8), two singlet methyls at δH 2.46 (3H, s, Me-10) and 2.48 (3H, s, Me-11), and a signal of hydroxyl proton at δH11.54. Analyses of the 13 C NMR spectra with the aid of the HSQC of 4 indicated the presence of 12 carbons including a carbonyl carbon at δC 160.52 (C=O), two aromatic methine at δC125.89 (C-8), 128.70 (C-5), two methyl carbons at δC19.43 (C-10), 20.08 (C-11), and seven quaternary aromatic carbons. Based on the chemical shift of four quaternary carbons at δC141.69 (C-4), 144.92 (C-7), 146.28 (C-2), 149.89 (C-3), it can be suggested that they linked to the nitrogen or oxygen atom. From the analysis of the masse and 1D-, 2D-NMR spectra and consulting the literature, compound 4 was confirmed to be 3,4-dihydroxy-6,7-dimethyl-quinoline-2 carboxylic [8]. The ESI-MS spectra of 5 showed the pseudo-molecular ion at m/z 155 [M+H] + .The 13 C- NMR and DEPT spectra of 5 showed the presence of seven carbons including four sp 3 methylene groups at δC23.28 (C-4), 29.36 (C-5), 46.30 (C-3), 47.00 (C-9), one sp 3 methine group at δC 59.84 (C-6), two carbonyl groups at δC166.44 (C-1), 171.98 (C-7). The chemical shifts of C-3 (δC 46.30), C-6 (δC 59.84) and C-9 (δC 47.00) suggested their linkage to oxygen and nitrogen atoms. The 1 H NMR spectrum of 5 showed signals of 9 protons at at the aliphatic region δH2.01-4.25. This observation suggested a cyclodipetide compound for 5. Comparison of NMR data revealed the structure of 5wich was identical tocyclo-(Pro-Gly) [9]. The ESI-MS spectra of 6 presented a base peak at m/z 195 [M-H] - . The 1 H-NMR spectrum showed the signals corresponding to a 1,2-disubstituted benzene ring δH 6.67 (2H, m, H-2+ H-4), 7.30 (1H, t, J= 7.5 Hz, H-3), 7.92 (1H, d, J= 7.5 Hz, H-1). Analysis of the 13 C-NMR and DEPT spectra of 6 indicated the presence of a carbonyl carbon at δC 173.0 (C = O), four aromatic methines at δC 135.00 (C-3), 132.14 (C-1), 116.80 (C-4), 116.49 (C-2) and two aromatic quaternary carbons at δC 109.78 (C-9a), 151.06 (C-4a). The chemical shifts of C-4a (C151.06) Secondary metabolites produced by marine actinomycete Streptomycessp. G246 63 suggested their linkages to oxygen atom. These observations suggested a structural symmetry in the structure of 6. Complete analysis of NMR, MS spectra and comparison with the reported data allowed determining the structure of 6 to be xanthone [10]. Compound 7 was isolated as a white solid. Its ESI mass spectrum showed the pseudo- molecular ion [M+H] + at m/z 284. The 1 H-NMR spectrum of 7 displayed signals of one singlet aromatic proton at δH 7.92(1H, s, H-8), a anomeric proton at δH 5.69 (1H, d, J = 6.0 Hz, H-1′) and a set of 5 protons of ribofuranose sugar moiety at ranging from δH3.52 to δH4.39. In the 1 H- 1 H COSY spectrum, the spin-spin coupling system of ribofuranose sugar moiety was observed (Figure 2). The 13 C-NMR and DEPT spectra of 7 showed the presence of 10 carbon atoms, including one sp 2 methine at δC 135.54(C-8), four sp 3 oxymethine at δC 70.35 (C-3′), 73.68 (C- 2′), 85.18 (C-4′), 86.35 (C-1′) and one sp3 oxymethylene at δC 61.39 (C-5′), and four quaternary carbons. The NMR spectra suggested that 7 was a purine nucleoside compound, forming from a guanine and a ribofuranose sugar unit. In the HMBC spectrum, the correlation of H-1′ (δH 5.69) with C-8 (δC 135.54), C-4 (δC 151.28), suggested that the guanine was attached to sugar unit via β-N9 bond (Figure 2). Detailed analysis of 2D NMR spectra, especially HMBC spectrum allowed determining the structure of 7 as guanosine [11]. Compound 8 was isolated as a white solid. The ESI-MS mass spectrum of 8 presented a base peak at m/z 252 [M+H] + . 1D NMR spectra ( 1 H-NMR and 13 C-NMR) of 8 displayed signals of 2'-deoxyribofuranoside moiety including threesp 3 oxymethine, one sp 3 oxymethylene and one sp 3 methylene. However, in the aromatic region, there are signals corresponding to an adenine moiety [δH8.20 (1H, s, H-8), 8.34 (1H, m, H-2); δC120.81 (C-5), 149.87 (C-4), 157.53 (C-6), 141.53 (C-8), 153.49 (C-2)]. This analyses suggested 8 being a nucleoside compound. Thus, analysis of MS and 1D NMR spectra determined 8 being 2′-deoxyadenosine [11]. Compound 9 was obtained as a white solid. The ESI-MS spectrum indicated the pseudo- molecular ion peak at m/z 268 [M+H] + . The signals on the 1 H-NMR spectrum of 9 were similar to those of 8, except for the appearance of an oxymethine (CH-2′) in 9 instead of a methylene in 8, and proton H-1′ appears as a doublet on the 1H NMR spectrum of 9. Whereas, this proton displayed a doublet of doublet in the 1 H NMR spectrum of 8. From the above analysis and reference, 9 has been identified as adenosine [12]. 4. CONCLUSIONS A comparison study was conducted to differentiate between solid and liquid fermentation technique for secondary metabolites production of a Vietnam marine actinobacteria (strain G246), in which, nine secondary metabolites including spirotry prostatins A (1), cyclo-(Pro-Met) (2), phenol A acid (3), 3,4-dihydroxy-6,7-dimethyl-quinoline-2-carboxylic (4), cyclo-(Pro-Gly) (5), xanthone (6), guanosine (7), 2′-deoxyadenosine (8), adenosine (9) were isolated from the solid state fermentation. Results revealed that compound 2 was the only one similarity between these fermentation techniques. Acknowledgements. The authors thank Prof. Do Cong Thung (Institute of Marine Environment and Resources, VAST) for marine sample collection and identification. This research was financially supported by the Fogarty International Center of the National Institutes of Health under Award Number D43TW010530. Vietnam Academy of Science and Technology (VAST) is gratefully acknowledged for financial support (Grant No. NVCC38.03/19-19). CRediT authorship contribution statement. D.T.Q.: Methodology, Formal analysis. D.T.M.H.: Formal analysis, Writing original draft Prepation, Review & Editing. T.V.H.: Formal analysis. T.B.N.: Formal Doan Thi Mai Huong, et al. 64 analysis. L.T.H.M.: Methodology, Formal analysis. V.T.Q.: Investigation, Formal analysis. N.T.H.A.: Formal analysis. B.T.B.: Funding acquisition, Methodology. P.V.C.: Project administration, Supervision, Formal analysis. Declaration of competing interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. REFERENCES 1. Labeda D. P. - Multilocus sequence analysis of phytopathogenic species of the genus Streptomyces, International Journal of Systematic and Evolutionary Microbiology 61 (10) (2010) 2525-2531. 2. 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