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MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY - CAO DUC TUAN PROJECT STUDY ON ANTIMICROBIAL SECONDARY METABOLITES ISOLATED FROM SELECTED MARINEDERIVED ACTINOBACTERIA STRAINS BELONG TO STREPTOMYCES GENUS Major: Organic Chemistry Code: 9.44.01.14 SUMMARY OF ORGANIC CHEMISTRY DOCTORAL THESIS HA NOI – 2020 This project was completed at Graduate University Science and Technology – Vietnam Academy of Science and Technology Scientific Advisor 1: Assoc Prof Doan Thi Mai Huong Scientific Advisor 2: Dr Le Thi Hong Minh Reviewer 1: … Reviewer 2: … Reviewer 3: … The thesis will be defended at Graduate University of Science and Technology – Vietnam Academy of Science and Technology, at: … The thesis can be found at: - The library of the Graduate University of Science and Technology, Vietnam Academy of Science and Technology - The National Library of Vietnam INTRODUCTION Basic of the thesis Natural resources have been used as drugs, additives and poisons for a long time (Marderosian, 1969) To date, natural products, most of them from terrestrial, play a major role in pharmaceutical industries (Harvey, 2000) Although, oceans accounted for about 70% of earth surface, proved to be very high biodiversity, researches on marine natural products only started recently (Fattorusso, 2012) There are over 30,000 marine natural products known to the public with diverse chemical structures and biological activities (Hu, 2011) Some reported marine natural products had structure similar to that of microorganism It suggested that microorganism may involve in the biosynthesis of these compounds or microorganism might be the production source (Molinski, 2009) Moreover, one of the major advantages of marine microbial research is that it only requires a small amount of natural collected marine samples Laboratory isolation, fermentation can help to produce enough material for further researches Vietnam, situated in the tropical Pacific region with high marine biodiversity, has enormous potential in marine resources Our government also supports the utilization of marine resources for social and economic development (Nguyễn Phú Trọng, 2018) Besides, Vietnam is one of a high antibiotic resistance prevalent country of the word (WHO, 2014) Therefore, development of new antimicrobial reagents domestically is urgently needed Based on those reasons, project “Study on antimicrobial secondary metabolites isolated from selected marine-derived actinobacteria strains belong to Streptomyces genus” was implemented Thesis aims Searching for antimicrobial secondary metabolites from actinobacteria isolated from Vietnam marine samples: - Isolation and screening for Vietnam antimicrobial marine-derived actinobacteria strains - Isolation and structure elucidation of compounds from promising isolated actinobacteria strains - Determination the antimicrobial activities of isolated compounds Main research activities Isolation and culture actinobacteria strains from collected marine samples; Screening the antimicrobial activities of isolated strains, select two promising strains for identification and fermentation (50L); Isolation of secondary metabolites from fermented strains; Structure elucidation of isolated compounds; Determination of antimicrobial activities of isolated compounds CHAPTER OVERVIEW 1.1 Marine biological resources 1.2 Actinomycetes 1.2.1 Classification of Actinomycetes 1.2.2 Streptomyces genus 1.2.3 Antibiotics from Streptomyces genus 1.3 Antimicrobial secondary metabolites from Streptomyces genus 1.3.1 World publications 1.3.1.1 Streptomyces sp isolated from marine sediments 1.3.1.2 Streptomyces isolated from marine invertebrates 1.3.1.3 Streptomyces isolated from other marine sources 1.3.1.4 Antimicrobial secondary metabolites developed by marine Streptomyces sp genome mining 1.3.2 Vietnam publications CHAPTER MATERIALS AND METHODS 2.1 Materials and equipment 2.2 Research Methods 2.2.1 Sample collection methods 2.2.2 Marine actinobacteria isolation method (Williams, 1965; Williams, 1971; Vũ Thị Minh Đức, 2001) 2.2.3 Marine actinobacteria purification and storage methods (Nguyễn Lân Dũng, 2003) 2.2.4 Marine actinobacteria activation and fermentation methods (Vũ Thị Minh Đức, 2001; Nguyễn Lân Dũng, 2003) 2.2.5 Culture broth extraction method (Carlson, 2014; Tanouye, 2015) 2.2.6 Actinobacteria identification methods (Holt, 1989; Sambrook, 1989; Weisburg, 1991; Li, 2007) 2.2.7 Actinobacteria fermentation (50 L) method (Basilio, 2003; Nguyễn Văn Cách, 2004) 2.2.8 Secondary metabolite isolation methods 2.2.9 Structural elucidation methods 2.2.10 Antimicrobial screening method (Hadacek, 2000) CHAPTER EXPERIMENT AND RESULTS 3.1 Sample collection results Using the described equipment and methods, 23 marine samples were collected, including: - 09 samples from the sea of Quang Binh (2 sediments, algae, soft corals and sponge) - 14 samples from Cu Lao Cham, Quang Nam (5 sediments, soft corals, sponge, echinoderms, sea-rabbits, snake tail and invertebrate) 3.2 Actinomycete isolation results Processing 23 marine samples collected at the sea of Quang Binh and Cu Lao Cham, Quang Nam led to the isolation of 35 actinobacteria strains with single colonies of unique morphology and color 3.3 Antimicrobial activities of isolated actinobacteria strains 35 isolated actinobacteria strains were cultured in small scale (500 mL) Their culture broths were extracted with EtOAc and screened for antimicrobial activities Obtain results (Table 4.1) showed that 24/35 isolated strain exhibited antimicrobial activities, of which, 4/35 stains were active against at least test microbial pathogens Two strain (G212 and G278), inhibited tested pathogens, representing two marine areas, were selected for further study 3.4 Identification of strain G212 and G278 3.4.1 Morphological properties of strain G212 and G278 3.4.2 Identification based on 16S rRNA gene sequence 3.4.2.1 Total DNA isolation 3.4.2.2 16S rRNA gene amplification 3.4.2.3 G212 and G278 16S rRNA gene sequences The 16S rRNA gene sequences of strain G212 and G278, were compared with GeneBank data, in combination with establishing neighbor joining tree Obtained results indicated that both strains (G212 and G278) had close relationship with members of genus Streptomyces The 16S rRNA gene sequences of strain G212 and G278 were registered in GeneBank with accession number of MF187963 and MF960781, respectively 3.5 Fermentation (50 L) of strain G212 and G278 Large scale fermentations were carried to afford fermentation broth (50L) of strain G212 and G278 3.6 Secondary metabolite isolation from Streptomyces sp G212 3.6.1 Culture broth extraction The EtOAc (EG212) and n-BuOH (BG212) extracts of 50 L fermentation broth of Streptomyces sp G212 was achieved with the mass of 2,2 g and 21,4 g, respectively 3.6.2 Secondary metabolite isolation from extracts of Streptomyces sp G212 3.6.2.1 Secondary metabolite isolation from EtOAc extract (EG212) From the EtOAc extract (EG212; 2,2 g), 10 compounds were isolated, including G212-1 (5 mg), G212-2 (6 mg), G212-3 (5 mg), G212-4 (7 mg), G212-5 (12 mg), G212-6 (7 mg), G212-7 (6 mg), G212-8 (8 mg), G212-9 (8 mg) and G212-10 (6 mg) 3.6.2.2 Secondary metabolite isolation from n-BuOH extract (BG212) From the n-BuOH extract (BG212; 21,4 g), compounds were isolated, including G212-11 (7 mg), G212-12 (13 mg), G212-13 (5 mg), G212-14 (6 mg), G212-15 (10 mg) and G212-16 (5 mg) 3.6.3 Physical parameter and spectral data of Streptomyces sp G212 isolated compounds 3.6.4 Total synthesis of compound G212-2 and G212-3 To confirm the structure of two new compound 2,4-dichlorophenyl 2,4dichlorobenzoate (G212-2) and 4,5-dihydroxy-7-methyl phthalide (G2123), total synthesis of compound G212-2 and G212-3 was achieved from the commercially available starting material as 2,4-dichlorobenzoyl chloride and 2,3-dimethoxybenzoic acid, respectively 3.6.4.1 Synthesis of 2,4-dichlorophenyl 2,4-dichlorobenzoate (G212-2 TH) 3.6.4.2 Synthesis of 4,5-dihydroxy-7-methyl phthalide (G212-3) and isomer G212-6’ 3.7 Secondary metabolite isolation from Streptomyces sp G278 3.7.1 Culture broth extraction The EtOAc (EG278) and n-BuOH (BG278) extracts of 50 L fermentation broth of Streptomyces sp G278 was achieved with the mass of 3,26 g and 22,4 g, respectively 3.7.2 Secondary metabolite isolation from extracts of Streptomyces sp G278 3.7.2.1 Secondary metabolite isolation from EtOAc extract (EG278) From the EtOAc extract (EG278; 3,26 g), compounds were isolated, including G278-1 (6 mg), G278-2 (7 mg), G278-3 (5 mg), G278-4 (10 mg), G278-5 (6 mg), G278-6 (6 mg) 3.7.2.2 Secondary metabolite isolation from n-BuOH extract (BG278) From the n-BuOH extract (BG278; 22,4 g), 10 compounds were isolated, including G278-7 (7 mg), G278-8 (6 mg), G278-9 (5 mg), G27810 (8 mg), G278-11 (6 mg), G278-12 (20 mg), G278-13 (6 mg), G278-14 (10 mg), G278-15 (8,5 mg), G278-16 (5 mg) 3.7.3 Physical parameter and spectral data of Streptomyces sp G278 isolated compounds 3.8 Antimicrobial activities of isolated compounds (Table 4.33 and Table 4.34) CHAPTER DISCUSSION OF RESULTS 4.1 Sample collection There were 23 collected marine sample, including samples from Quang Binh and 14 samples from Cu Lao Cham, Quang Nam The number and type of marine samples collected in each area are different, might be reflecting the differences in local biodiversity at the time of sample collection 4.2 Actinomycete isolation From marine samples collected at the sea of Quang Binh and 14 marine samples collected at the sea of Cu Lao Cham, Quang Nam, 15 and 20 actinobacteria strains were isolated, respectively 4.3 Antimicrobial activities of isolated actinobacteria strains Antimicrobial screening results of EtOAc extract of 35 isolated actinobacteria strains (Table 4.1) revealed that 24/35 (68,6 %) strains exhibited antimicrobial activities Of which, 4/35 (11,4 %) strains active against at least tested microorganisms; 11/35 (31,4 %) strongly active against Candida albicans (MIC – 64 µg/mL) Besides, there were 15/35 (42,8%) strain inhibited the growth of Gram positive bacteria while only 6/35 (17,1%) strains inhibited the growth of Gram negative bacteria Table 4.1 The MIC (µg/mL) of isolated actinobacteria strains (* Only active strains were presented) Minimum Inhibitory Concentration MIC (µg/mL) No Strain (*) Gram positive E faecalis S aureus B cereus Gram negative E coli P aeruginosa S enterica fungus C.albicans ATCC29212 ATCC25923 ATCC14579 ATCC25922 ATCC27853 ATCC13076 ATCC10231 15 actinobacteria strains isolated from Quang Binh sea G183 128 G193 G196 64 G197 128 G202 128 G207 128 G212 128 128 64 256 - 64 - 256 9 10 11 12 13 14 15 G214 128 G216 256 20 actinobacteria strains isolated from Cu Lao Cham, Quang Nam sea G274 256 G275 G276 256 G277 256 G278 256 32 16 16 G280 256 256 16 32 32 G283 128 G284 256 256 G285 32 G288 32 G289 32 G290 32 16 16 G291 128 G292 32 G293 256 S 256 256 128 32 256 128 C 32 (S: Streptomycine; C: Cyclohexamide; (-): MIC > 256 µg/mL) 4.4 Identification of strain G212 and G278 The 16S rRNA gene sequence of strain G212 were 99,57% similar to that of Streptomyces caelestis JS-5 (GeneBank accession no EU124773) and 99,64% similar to Streptomyces caelestis JS-4 (GeneBank accession no EU124772) The 16S rRNA gene sequence of strain G278 were 99,86% similar to that of Streptomyces sp R1 (GeneBank accession no MK757961) 4.5 Fermentation (50 L) of strain G212 and G278 The large-scale fermentations (50 L) of strain G212 and G278 were successfully performed to afford required materials for next research step 4.6 Structure elucidation of secondary metabolites isolated from Streptomyces sp G212 From the culture broth of Streptomyces sp G212, the isolation and structure elucidation of 16 compounds was achieved (Figure 4.31), including two new compounds (G212-2 and G212-3) and one compound isolated first time from nature (G212-1) These isolated compounds were identified as polyethylene terephthalate: ethylene terephthalate cyclic trimer (G212-1), dichlorophenyl derivative: 2,4-dichlorophenyl 2,4-dichlorobenzoate (G2122), phthalite derivative: 4,5-dihydroxy-7-methyl phthalide (G212-3), antibiotics: germicidine A (G212-4), germicidine B (G212-5), benzopyridine derivative: 3,4-dihydroxy-6,7-dimethyl-quinolin-2carboxylic (G212-9), cyclopeptide: cyclo-(Pro-Val) (G212-10), cyclo(Pro-Tyr) (G212-11), cyclo-(Leu-trans-4-hydroxy-Pro) (G212-12), nucleic acid derivative: 2’-deoxythymidine (G212-7), 2’-deoxyuridine (G212-8), indole: N-[2-(1H-indol-3-yl)-2-oxo-ethyl] acetamide (G21213), indole-3-carboxylic acid (G212-14) and 5-hydroxymethyl-4-hydroxy2,4-dimethyl-2-cyclopentenone (G212-6), 1H-pyrrole-2-carboxylic acid (G212-15), 2-phenylacetic acid (G212-16) Structure of two new compounds, G212-2 and G212-3, were confirmed by total synthesis Figure 4.31 Compounds isolated from G212 10 of unsaturation and the molecular formula (C30H24O12) established from the HR-ESI mass spectrum, assigned a trimeric cyclic structure for compound G212-1 The structure of G212-1 was confirmed by X-ray diffraction analysis (Figure 4.4) Therefore, compound G212-1 was identified as ethylene terephthalate cyclic trimer This compound was previously reported as a synthetic derivative (Kint et al., 2003) 4.6.2 2,4-dichlorophenyl 2,4-dichlorobenzoate (G212-2) Compound G212-2 was isolated as a white solid Its HR-ESI mass spectrum showed the proton adduct molecular ion [M+H]+ at m/z 334.9194 (with chlorine isotopic pattern at m/z 334.9194, 336.9167, 338.9141 and 340.9117), which together with 13 C-NMR data is consistent with a molecular formula of C13H6Cl4O2 The H-NMR spectrum of compound G212-2 displayed signals corresponding to two ABX aromatic ring systems [A-ring: H 7.40 (dd, J = 2.0, 8.5 Hz, H-5), 7.57 (d, J = 2.5, H-3), 8.11 (d, J = 8.5, H-6), and B-ring: H 7.23 (d, J = 8.5, H-6′), 7.32 (dd, J = 2.5, 8.5, H-5′), 7.50 (d, J = 2.5, H-3′)] which were supported by COSY spectrum data (Figure 4.10) Analysis of the 13C-NMR spectrum with the aid of the HSQC experiment of G212-2 indicated the presence of 13 carbons, including carbonyl group at C 161.7, methines at C 124.6 (C-5′), 127.3 (C-6), 128.1 (C-6′), 130.3 (C-3′), 131.5 (C-3) and 133.3 (C-5), and quaternary carbons at C 126.4 (C-1), 127.9 (C-2′), 132.4 (C-4′), 136.2 (C-4), 139.7 (C-2) and 145.5 (C-1′) In the HMBC spectrum (Figure 4.10), the correlation of H-6 (H 8.11) with the carbonyl carbon (C 161.7) assigned the connection of the carbonyl group to the A-ring The carbon chemical shift of C1′ (C 145.5) suggested its linkage to oxygen Considering the molecular formula of C13H6Cl4O2 established above, the structure of G212-2 was identified as 2,4dichlorophenyl 2,4-dichlorobenzoate (Figure 4.31) Since the ester linkage of the A- and B-ring could not be resolved by HMBC data analysis, the structure of G212-2 was then confirmed by a simple synthesis step which was achieved by reaction of 2,4-dichloro-benzoyl chloride and 2,4-dichlorophenol (Figure 4.11) The reaction was carried-out at oC to room temperature in the presence of Et3N The 1H NMR spectrum of the synthetic sample was identical with that of the natural compound G212-2 and this is a new compound 11 Table 4.3 NMR data of G212-2 and synthetic compound G212-TH G212-2 G212-2 TH C a,b a,c a,b,# δH mult (J, Hz) δC δH mult (J, Hz) 126.4 139.7 7.57 d (2.5) 131.5 7.56 d (2.0) 136.2 7.40 dd (2.0; 8.5) 133.3 7.39 dd (2.0; 8.5) 8.11 d (8.5) 127.3 8.11 d (8.5) 161.7 1' 145.5 2' 127.9 3' 7.50 d (2.5) 130.3 7.50 d (2.5) 4' 132.4 124.6 5' 7.32 dd (2.5; 8.5) 7.31 dd (2.5; 8.5) 6' 7.23 d (8.5) 128.1 7.23 d (8.5) Measured in: aCDCl3, b500 MHZ, c125 MHz, #H of G212-2 TH Figure 4.10 Main COSY and HMBC correlations of G212-2 Figure 4.11 Reaction scheme for G212-2 TH 4.6.3 4,5-dihydroxy-7-methyl phthalide (G212-3) Compound G212-3 was isolated as a white solid The HR-ESI mass spectrum showed the proton adduct molecular ion [M+H]+ at m/z 181.0496 which together with 13C-NMR data, suggested a molecular formula of C9H8O4 for G212-3 Its IR absorptions implied the presence of hydroxyl groups (3475 cm-1) and carbonyl functionality (1717 cm-1) 12 Table 4.4 NMR data of G212-3 and synthetic compound G212-3 TH G212-3 G212-3 TH C a,b a,c a,b,# δH mult (J, Hz) δC δH mult (J, Hz) 170.9 5.13 s 66.7 5.13 s 3a 134.7 137.1 150.6 6.72 s 118.4 6.72 s 130.1 7a 113.5 2.38 s 16.0 2.38 s Measured in aDMSO-d6, b500 MHZ, c125 MHz, #H of G212-3 TH The 1D NMR (1H and 13C) spectra of G212-3 showed one methyl group (H 2.38, C 16.0), one methylene (H 5.13, C 66.7), one sp2 methine (H 6.72, C 118.4), five aromatic quaternary carbons (C 113.5, 130.1, 134.7, 137.1 and 150.6), and one carbonyl carbon (C 170.9) The six degrees of unsaturation were thus assigned to compound G212-3 The chemical shifts of CH2-3, C-4 and C-5 suggested their linkage to oxygen (Table 1) Intensive analyses of the 2D NMR spectral data, especially the HMBC spectrum (Figure 4.16) indicated that compound G212-3 was a phthalide derivative However, the protons of CH2-3 (H 5.13) were correlated to both C-4 (C 137.1) and C-7 (C 130.1) in the HMBC spectrum Thus, one of these crosspeaks should be a four-bond long-range correlation This observation suggested two structural possibilities for compound G212-3 (Figure 4.12) To confirm the structure of G212-3 and to obtain enough quantity for biological assay, a total synthesis of G212-3 and its isomer G212-6’, was performed As indicated in Figure 4.17, the commercially available substance 2,3-dimethoxy benzoic acid was used as starting material which was converted into G212-3 through major steps Besides, to differentiate the structure of G212-4′ and G212-5′, compound G212-4′ was also one step synthesized from the halogenated G212-1′ 13 Figure 4.12 Two structural possibilities for compound G212-3 Figure 4.16 Main HMBC correlations of G212-3 (a) HCHO, concentrated HCl, 60-70 oC, 80%; (b) LiAlH4, THF, 72%; (c) i, KMnO4, NaOH, H2O; ii, Ac2O, 55% steps; (d) LiAlH4, THF, 77%; (e) H2, Pd/C, THF, 97%; (f) BBr3, CH2Cl2, oC - RT, 89%; (g) BBr3, CH2Cl2, 0oC-RT, 87% Figure 4.17 Synthesis scheme of G212-3 TH and isomer G212-6′ Synthesis of 4-(chloromethyl)-6,7-dimethoxyisobenzofuran-1(3H)-one (G212-1′) 14 The commercially available 2,3-dimethoxy benzoic acid was turned to phthalide derivative G212-1′ with 80 % yield, using concentrated HCl and paraformaldehyde at 60 - 70 oC, reaction time 3h (Bhattacharjee, 1980) The 1H-NMR and 13C-NMR spectra of G212-1′ gave signals of a carbonyl group at C 168.1; a methine group (C 119.8; H 7.20); methoxy groups (C 57.1; 62.5, H 3.93; 4.11), methylene groups and quaternary carbons Those NMR data confirmed the structure of the phthalide derivative G212-1′ Synthesis of 3,4-dimethoxy-6-methyl-1,2-phenylene dimethanol (G2122′): In the next step, compound G212-1′was reduced by LiAlH4 in THF at reflux to afford the alcohol G212-2′ in 72% yield (Ying, 2011) In comparison with G212-1′, the 1H-NMR and 13C-NMR data of G212-2′ indicated the absent of a carbonyl group and additional signals of a methyl group at C 19.6, H 2.39 These NMR suggested that the lactone ring in compound G212-2′ was open to give an alcohol Synthesis of 4,5-dimethoxy-7-methylisobenzofuran-1,3-dione (G212-3′) The synthesis of G212-3′ was carried in a 2-step procedure First, G212-2′ was oxidized by KMnO4, yielding the acidic product After that, the crude acidic product was refluxed with mL of Ac2O in 30 minutes to give phthalic anhydride derivative G212-3′ in 55% yield The signals appeared in 1H-NMR and 13C-NMR spectra of G212-3′ were close to those of G212-2′, accept for the presence of two carbonyl groups at C 162.6 and 160.9 instead of two methylene groups These NMR data led to the structure establishment of compound G212-3′ 15 Synthesis of G212-4′ and G212-5′ Compound G212-3′ was then treated with LiAlH4 to provide a mixture of two isomers G212-4′ and G212-5′ with a ratio of 1/2, respectively The two isomers G212-4′ and G212-5′ were successfully separated by column chromatography (silica gel; n-hexane/ EtOAc gradient) and their structures were confirmed by NMR data analysis Both NMR spectra of compound G212-4′ and G212-5′ showed signals of a methyl group, a carbonyl, a methin group, methoxy groups and quaternary carbons The HMBC spectra of G212-5′ (Figure 4.19) showed the correlations between protons of CH3-10 group with carbon C-7a, the carbon directly connecting to carbonyl group (C 116.1) This correlation suggested the structure of compound G212-5′ However, the HMBC spectra of G2125′ also indicated the correlations between CH3-10 protons with C-3a and C4 Hence, in order to support for the structural distinction between the two isomers G212-4′ and G212-5′, compound G212-4′ was also synthesized from the halogenated compound G212-1′ by catalytic hydrogenation of Pd/C The NMR data of compound G212-4′ synthesized from two different routes are identical, allowing the structure determination of two isomers G212-4′ and G212-5′ Figure 4.19 Main HMBC correlations of G212-5′ 16 C 3a 7a 10 Table 4.5 NMR data of G212-4′ and G212-5′ G212-4′ G212-4′ G212-5′ Syn from Syn from G212-3′ G212-1′ a,b δHa,b δ δHa,b H δCa,c δCa,c mult (J, Hz) mult (J, Hz) mult (J, Hz) 169.0 170.8 5.12 s 68.0 5.12 s 5.24 66.7 137.8 138.8 120.6 140.7 7.02 s 117.6 7.02 s 156.0 152.5 6.81 s 116.1 146.5 136.0 126.8 115.3 4.05 s 62.3 4.05 s 3.90 s 56.3 3.90 s 57.0 3.90 s 3.95 s 60.4 2.26 s 17.2 2.26 s 2.63 17.1 Measured in aCDCl3, b500 MHz, c125 MHz Synthesis of G212-3 and G212-6′ Finally, exposure of G212-4′ and G212-5′ to BBr3 in THF afforded compounds G212-6′ and G212-3 TH, respectively, 87-89 % yield The NMR datal of G212-3 (natural) and G212-3 TH (synthesized) were identical Thus, compound G212-3 was confirmed to be 4,5-dihydroxy-7methyl phthalide G212-3 and its synthetic isomer (G212-6′) were both new compounds 17 C 3a 7a OH-6 OH-7 Table 4.6 NMR data of G212-3 TH and G212-6′ G212-3 TH G212-6′ a,b a,c a,b δH mult (J, Hz) δC δH mult (J, Hz) 170.9 5.13 s 66.7 5.11 s 134.7 137.1 150.6 6.92 s 6.72 s 113.5 118.4 111.5 2.38 16.0 2.08 s 9.32 s 9.67 s δCa,c 169.8 68.1 136.4 123.1 121.8 142.7 145.3 111.5 16.4 - Measured in aDMSO-d6, b500 MHZ, c125 MHz 4.7 Structure elucidation of secondary metabolites isolated from Streptomyces sp G212 Figure 4.49 Isolated compounds from strain G278 18 From the culture broth of Streptomyces sp G278, the isolation and structure elucidation of 16 compounds was achieved (Figure 4.49), including two compound isolated first time from nature G278-15 and G278-16 Among them, there were cyclodipeptides: cyclo-(Pro-Gly) (G278-1), cyclo-(Pro-Leu) (G278-2), cyclo-(Pro-Phe) (G278-3), cyclo (Pro-Tyr) (G278-4), cyclo-(Leu-Tyr) (G278-5), cyclo-(Pro-Trp) (G278-6), one indol compound 1H-indole-3-ethanol (G278-7), coumarin compound: scopoletin (G278-8), nucleoside: compound adenosine (G278-11), dioxan compound: 2-((-5-methyl-1,4-dioxan-2-yl)methoxy)ethanol (G27812) and other phenolic compounds: benzyl salicylate (G278-9), Nphenylnaphthalen-2-amine (G278-10), N-(4-hydroxyphenylethyl)acetamide (G278-13), 2,4-dichlorophenyl 2,4-dichlorobenzoate (G278-14), 2,5-bis(5tert-butyl-2-benzoxazolyl)thiophene (G278-15), 3-hydroxyl-2( methylpyridine G278-16) Structure characterizations of G278-15 and G278-16 are presented below 4.7.1 2,5-Bis(5-tert-butyl-2-benzoxazolyl)thiophene (G278-15) Compound G278-15 was isolated as a white solid HR-ESI mass spectrum of G278-15 presented a pseudo-molecular ion peak at m/z 431.1785 [M+H]+, together with 13C NMR data which were consistent with a molecular formula of C26H26N2O2S Fifteen degrees of unsaturation were thus assigned for G278-15 Its IR spectrum showed absorbance of C=N (1635, 1581 cm-1) and C-O (1266, 1195 cm-1) functionalities Moreover, the presence of sulfur atom in the structure of G278-15was supported by the IR absorption band of C-S at νmax 715 cm-1 In the 1H-NMR spectrum, compound G278-15 displayed signals of an ABX system at δH 7.54 (1H, dd, J = 2.0, 8.0 Hz, H-5), 7.72 (1H, d, J = 8.0 Hz, H-4 ), 7.81 (1H, d, J = 2.0 Hz, H-7 ), a singlet aromatic proton at 8.06 (1H, s, H-3’), and three methyl groups at δH 1.37 (9H, s, x CH3) The 13C-NMR and DEPT spectra of G278-15 indicated the presence of 13 carbon signals Analysis of the HMBC spectrum (Figure 4.38) determined the connection of the tert-butyl group at C-6 of the ABX aromatic system by cross-peaks of C-6 (δC 148.3) with three CH3 protons at δH 1.37, H-4, H-5, and H-7 Additionally, the proton H-3’ (δH 8.06) correlated with the two remaining 19 carbons C-2 (δC 157.5) and C-2’ (δC 132.4), assigning the connection of C-3’/C-2’/C-2 The chemical shifts of C-7a (δC 148.6) and C-3a (δC 141.3) suggested their linkage to oxygen and nitrogen, respectively This observation, together with the chemical shift of C-2 (157.5 ppm) suggested an 1,3-oxazine ring system in the structure of G278-15 These analyses indicated a sub-structure with a formula of C13H13NO for G27815 Taking into account the molecular formula C26H26N2O2S established from the HR-ESI-MS, compound G278-15thus possessed a symmetric structure in which the two sub-structural units C13H13NO were connected each other via C-3–C-3’ and C-2’–S–C-2’’ linkages (Figure S1) Complete analysis of 2D-NMR spectra indicated that compound G27815 was 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene This compound, previously synthesized and used as a fluorescent brightening agent (Li, et al 2008), was reported here for the first time from a natural source Table 4.31 NMR data of compound G278-15 G278-15 C a,b δH mult (J, Hz) δCa,c 157.5 3a 141.3 7.72 d (8.0) 110.3 7.54 dd (2.0; 8.0) 123.9 148.3 7.81 d (2.0) 116.3 7a 148.6 34.8 1.37 s 31.5 2′ 132.4 3′ 8.06 s 131.3 Measured in aDMSO-d6, b500 MHZ, c125 MHz Figure 4.38 Main HMBC correlations of compound G278-15 20 4.7.2 3-hydroxy-2-methylpyridine (G278-16) Compound G278-16 was obtained as white solid Its positive HR-ESI-MS showed the proton adduct ion [M+H]+ at m/z 110.0609 (calcd for C6H7NO, 110.0606) which, together with 13C NMR data, is consistent with the molecular formula of C6H6NO The 1H-NMR spectrum of G278-16 showed the signals of three aromatic protons at δH 7.01 (1H, dd, J = 5.0, 8.0 Hz, H5), 7.09 (1H, d, J = 8.0 Hz, H-4), 7.86 (1H, d, J = 5.0 Hz, H-6) and methyl protons at δH 2.30 (3H, s) The 13C NMR and DEPT spectra of G278-16 displayed the presence of the groups observed above from the 1H NMR spectrum, with additional two aromatic quaternary carbons δC 147.7 (C-2) and 154.0 (C-3) The assignment of carbon signals corresponding to each proton signal was achieved by a HSQC experiment The proton coupling constants of H-4, H-5 and H-6, together with the chemical shifts of C-2 and C-6 suggested the presence of a pyridine ring This suggestion was confirmed by the HMBC spectrum analysis (Figure 4.48), in which the cross-peaks of methyl protons at δH 2.30 with C-2 and C-3, H-5 with C-3, and H-6 and H-4 with C-2 were observed The chemical shift of C-3 (δC 154.0) suggested its linkage to oxygen Thus, compound G278-16 was identified as 3-hydroxyl-2-methylpyridine This compound has been previously synthesized (Jida and Ollivier, 2008), however, this is the first report of G278-16 from a natural source Table 4.32 NMR data of compound G278-16 and reference compound G278-16 3-hydroxy-2-methylpyridine C a,c a,d δH mlult (J, Hz) δC δH b,e# mlult (J, Hz) δC b,f# 147.7 144.7 154.0 146.3 7.09 d (8.0) 122.6 7.15 dd (1.2; 8.4) 122.3 7.01 dd (5.0; 8.0) 123.2 7.06 dd (4.7; 8.4) 122.4 7.86 d (5.0) 139.3 8.05 dd (1.2; 4.7) 139.7 2.30 s 18.4 2,53 m 18.4 Measured in aCD3OD, bCDCl3, c500 MHZ, d125 MHz, e300 MHZ, f63 MHz, #H of reference compound (Jida and Ollivier, 2008) Figure 4.48 Main HMBC correlations of compound G278-16 21 4.8 Antimicrobial activities of isolated compounds All isolated and synthesized compounds were screened for antimicrobial activities Obtained results indicated that 20/30 isolated compounds and synthesized compounds exhibited antimicrobial activities (Table 4.33 and Table 4.34) Table 4.33 and Table 4.34 MIC (µg/mL) values of isolated and synthesized compounds (* Only active compounds were reported) Minimum Inhibitory Concentration - MIC (µg/mL) N Comp (*) Gram positive E faecalis S aureus B cereus Gram negative E coli P aeruginosa S enterica fungus C.albicans ATCC29212 ATCC25923 ATCC14579 ATCC25922 ATCC27853 ATCC13076 ATCC10231 10 11 12 13 14 15 17 18 19 20 Natural isolated compound from G212 and G278 G212-1 64 G212-2 256 G278-14 G212-3 64 128 128 256 G212-4 16 64 32 64 G212-5 64 128 64 G212-6 32 64 64 128 128 G212-7 32 32 64 32 32 G212-8 16 32 32 64 G212-9 128 32 64 G212-15 64 256 G212-16 32 G278-5 32 G278-6 128 G278-8 64 G278-9 256 G278-10 128 256 256 128 G278-12 32 G278-15 64 G278-16 256 256 64 256 Synthesized compounds G212-4' 64 64 128 128 64 128 G212-5' 128 64 128 32 G212-6' 128 64 32 64 32 S 256 256 128 32 256 128 C (S: Streptomycine; C: Cyclohexamide; (-): MIC > 256 µg/mL) 64 128 64 64 32 32 128 64 64 128 32 32 22 CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS From 23 marine samples collected the sea of Quang Binh and Cu Lao Cham, Quang Nam, 35 actinobacteria strains were isolated The antimicrobial activities of 35 isolated strains were tested against a panel of clinical important pathogens, of which, 24/35 strains (68,6 %) exhibited inhibition activities and 4/35 strains (11,4 %) inhibited the growth of at least tested micro-organisms Strain G212 and G278, active against 5/7 tested microorganisms, was identified belonging to Streptomyces genus, their 16S rRNA gene sequences were registered with GenBank access code of MF187963 and MF960781, respectively There were 32 secondary metabolites (including 30 different compounds) isolated from trains G212 and G278 Of which, two compounds 2,4-dichlorophenyl 2,4-dichlorobenzoate (G212-2) and 4,5-dihydroxy-7-methyl phthalide (G212-3) were new; and there compounds ethylene terephthalate cyclic trimer (G212-1), 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene (G27815) and 3-hydroxy-2-methylpyridine (G278-16) were reported for the first time from nature To confirm the structure of two new compound G212-2 and G2123, total synthesis was achieved These syntheses not only produced enough quantities of G212-2 and G212-3 for their biological screening but also allowed to obtain three new derivatives of G212-3 The structure of the first time from nature compound G212-1 was confirmed by X-ray Testing isolated compounds against a panel of microbial pathogens revealed that 20/30 isolates shown inhibition activities against tested pathogens Three synthesized analogues of compound G212-3 exhibited antimicrobial activites While synthesized isomer G212-6’ strongly inhibited fungus C albicans and Gram negative bacteria P aeruginosa (MIC = 32 µg/ml), the natural isomer G212-3 was not active against C albicans 23 RECOMMENDATIONS Further biological characterizations should be conducted for isolated compounds that exhibited antimicrobial activities Further study on un-touched isolated antimicrobial actinobacteria strains should be carried The scope of this project should also expanded to other marine areas for identification of marine microorganisms with strong biological active secondary metabolites NEW FINDINGS OF THE THESIS 35 actinobacteria strains were isolated from 23 marine samples collected in the sea of Quang Binh and Cu Lao Cham - Quang Nam, Viet Nam Their antimicrobial activities were tested against a panel of clinical important pathogens, of which, 24/35 strains (68,6 %) exhibited inhibition activities and 4/35 strains (11,4 %) inhibited the growth of at least tested micro-organisms Strain G212 and G278, active against 5/7 tested microorganisms, was identified belonging to Streptomyces genus, their 16S rRNA gene sequences were resisted with GenBank acess code: MF187963 and MF960781, respectively There were 32 secondary metabolites (including 30 different compounds) isolated from trains G212 and G278 Of which, two compounds 2,4-dichlorophenyl 2,4-dichlorobenzoate (G212-2) and 4,5-dihydroxy-7-methyl phthalide (G212-3) were new and there compounds ethylene terephthalate cyclic trimer (G212-1), 2,5-bis(5-tert-butyl-2-benzoxazolyl)thiophene (G278-15) and 3-hydroxy-2-methylpyridine (G278-16) were reported for the first time from nature To confirm the structure of two new compound G212-2 and G212-3, total synthesis was achieved These syntheses not only produced enough quantities of G212-2 and G212-3 for their biological screening but also allowed to obtain three new derivatives of G212-3 Structure of compound G212-1 was also confirmed by X-ray experiment All obtained compounds were screened for their antimicrobial activities Results revealed that 20/30 isolates and synthetic compounds shown inhibition activities against tested microbial pathogens 24 PUBLICATIONS WITH THE THESIS Duc-Tuan Cao, Thuy-Linh Nguyen, Van-Hieu Tran, Huong Doan-ThiMai, Quyen Vu-Thi, Mai-Anh Nguyen, Hong-Minh Le-Thi, Van-Minh Chau and Van-Cuong Pham, Synthesis, Structure and Antimicrobial Activity of Novel Metabolites from a Marine Actinomycete in Vietnam’s East Sea, 2019 Natural Product Communication, 14 (1): 121-124 Duc Tuan Cao, Van Hieu Tran, Van Nam Vu, Huong Doan Thi Mai, Thi Hong Minh Le, Thi Quyen Vu, Hung Huy Nguyen, Van Minh Chau, Van Cuong Pham, Antimicrobial Metabolites from a Marine-Derived Actinomycete Streptomyces sp G278, 2019 Natural Product Research, 33:22, 3223-3230 Cao Duc Tuan, Le Thi Hong Minh, Vu Thi Quyen, Nguyen Mai Anh, Doan Thi Mai Huong, Chau Van Minh, Pham Van Cuong, Identification and Antimicrobial Activity of Actinomycetes Strains isolated from samples collected in the coastal area Hue, Da Nang and Quang Nam Provinces, Vietnam, 2017 Tạp chí Cơng nghệ Sinh học, 15 (4): 737-744 Cao Duc Tuan, Vu Van Nam, Tran Van Hieu, Doan Thi Mai Huong, Vu Thi Quyen, Nguyen Mai Anh, Le Thi Hong Minh, Brian T Murphy, Nguyen Quoc Vuong, Pham Van Cuong, Secondary metabolites from marine actinomycete Streptomyces sp G212, 2017 Tạp chí Hóa học, 55 (6e): 8589 Cao Duc Tuan, Tran Van Hieu, Doan Thi Mai Huong, Le Thi Hong Minh, Vu Thi Quyen, Nguyen Mai Anh, Brian Murphy, Nguyen Quoc Vuong, Pham Van Cuong, Secondary metabolites produced by marine actinomycette Streptomyces sp G278 2017, Tạp chí Hóa học, 55 (6e): 145-149 Cao Duc Danh, Cao Duc Tuan, Vu Thi Quyen, Nguyen Mai Anh, Doan Thi Mai Huong, Pham Van Cuong, Chau Van Minh, Le Thi Hong Minh, Identification and antimicrobial activity of actinomycetes strains isolated from marine samples in the coastal area of Thanh Hoa – Quang Binh – Quang Tri, 2018 Vietnam Journal of Science and Technology, 56 (4): 424433 ... Technology – Vietnam Academy of Science and Technology Scientific Advisor 1: Assoc Prof Doan Thi Mai Huong Scientific Advisor 2: Dr Le Thi Hong Minh Reviewer 1: … Reviewer 2: … Reviewer 3: … The... to Streptomyces genus” was implemented Thesis aims Searching for antimicrobial secondary metabolites from actinobacteria isolated from Vietnam marine samples: - Isolation and screening for Vietnam... activities of isolated compounds CHAPTER OVERVIEW 1.1 Marine biological resources 1.2 Actinomycetes 1.2.1 Classification of Actinomycetes 1.2.2 Streptomyces genus 1.2.3 Antibiotics from Streptomyces
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