Study on antimicrobial secondary metabolites isolated from selected marinederived actinobacteria strains belong to streptomyces genus

From 23 marine samples collected the sea of Quang Binh and Cu Lao

Cham, Quang Nam, 35 actinobacteria strains were isolated.

2. 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 4 tested micro-organisms.

3. 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.

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212 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), 6 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). 5 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 2,4- dichlorobenzoate (G212-2) and 4,5-dihydroxy-7-methyl phthalide (G212- 3), 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), 6 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), G278- 10 (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) 6 CHAPTER 4. DISCUSSION OF RESULTS 4.1. Sample collection There were 23 collected marine sample, including 9 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 9 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 4 tested microorganisms; 11/35 (31,4 %) strongly active against Candida albicans (MIC 2 – 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) No Strain (*) Minimum Inhibitory Concentration MIC (µg/mL) Gram positive Gram negative fungus E. faecalis ATCC29212 S. aureus ATCC25923 B. cereus ATCC14579 E. coli ATCC25922 P. aeruginosa ATCC27853 S. enterica ATCC13076 C.albicans ATCC10231 15 actinobacteria strains isolated from Quang Binh sea 1 G183 - - 128 - - - - 2 G193 - - - - - - 64 3 G196 - - - 64 - - - 4 G197 - - 128 - - - - 5 G202 - - - 128 - - - 6 G207 128 - - - - - - 7 G212 128 - 128 - 64 256 256 7 8 G214 - - 128 - - - - 9 G216 - 256 - - - - - 20 actinobacteria strains isolated from Cu Lao Cham, Quang Nam sea 1 G274 256 - - - - - - 2 G275 - - - - - - - 3 G276 256 - - - - - 8 4 G277 - - - - - - 256 5 G278 256 32 - 16 - 16 2 6 G280 256 - 256 16 32 - 32 7 G283 128 - - - - - - 8 G284 256 256 - - - - - 9 G285 - - - - - - 32 10 G288 - - - - - - 32 11 G289 - - - - - - 32 12 G290 - - 32 - 16 8 16 13 G291 - 128 - - - - - 14 G292 - - - - - - 32 15 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 8 two new compounds (G212-2 and G212-3) and one compound isolated first time from nature (G212-1). These isolated compounds were identified as 1 polyethylene terephthalate: ethylene terephthalate cyclic trimer (G212-1), 1 dichlorophenyl derivative: 2,4-dichlorophenyl 2,4-dichlorobenzoate (G212- 2), 1 phthalite derivative: 4,5-dihydroxy-7-methyl phthalide (G212-3), 2 antibiotics: germicidine A (G212-4), germicidine B (G212-5), 1 benzopyridine derivative: 3,4-dihydroxy-6,7-dimethyl-quinolin-2- carboxylic (G212-9), 3 cyclopeptide: cyclo-(Pro-Val) (G212-10), cyclo- (Pro-Tyr) (G212-11), cyclo-(Leu-trans-4-hydroxy-Pro) (G212-12), 2 nucleic acid derivative: 2’-deoxythymidine (G212-7), 2’-deoxyuridine (G212-8), 2 indole: N-[2-(1H-indol-3-yl)-2-oxo-ethyl] acetamide (G212- 13), indole-3-carboxylic acid (G212-14) and 5-hydroxymethyl-4-hydroxy- 2,4-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 9 4.6.1. Ethylene terephthalate cyclic trimer (G212-1) Compound G212-1 was obtained as a microcrystalline material and its molecular formula C30H24O12 was determined from HR-ESI-MS m/z 577.1349 [M+H]+ (calcd. for C30H25O12 m/z 577.1346). Thus, nineteen degrees of unsaturation were assigned for G212-1. The IR spectrum indicated the presence of ester groups (1729 cm-1), aromatic groups (1578 and 1457 cm-1). The 1H-NMR spectrum of 3 showed a only two singlet signals at H 8.06, and at H 4.66. These two peaks were equally integrated. Analysis of the 13C-NMR and DEPT spectra of 3 revealed the resonances of a carbonyl at C 165.3, a methylene carbon at C 62.7, an aromatic methine carbon at C 129.7, and a quaternary carbon at C 133.8. The chemical shifts of the methylene (H 4.66, C 62.7) suggested their linkages to oxygen atom. These observations suggested a structural symmetry in the structure of G212-1. Table 4.2. NMR data of G212-1 and reference compound. C G212-1 Poly(ethylene terephthalate) δHa,c mult. (J, Hz) δCa,d δC a,e,# 1 - 165.3 168 2 - 133.8 134 3 8,10 s 129.7 130 4 4,69 s 62.7 64 Measured in aCDCl3, bDMSO-d6, c500 MHZ, d125 MHz; e75 MHz, # C Poly(ethylene terephthalate) (Kint, 2003). Figure 4.4. HMBC correlations and X-ray structure of G212-1 These spectral features were similar to those of polyethylene terephthalate (Backson et al. 1995, Štokr et al. 1982). Taking into account nineteen degrees 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 13C-NMR data is consistent with a molecular formula of C13H6Cl4O2. The 1H-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 1 carbonyl group at C 161.7, 6 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 6 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 C- 1′ (C 145.5) suggested its linkage to oxygen. Considering the molecular formula of C13H6Cl4O2 established above, the structure of G212-2 was identified as 2,4- dichlorophenyl 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 0 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 C G212-2 G212-2 TH δHa,b mult. (J, Hz) δCa,c δH a,b,# mult. (J, Hz) 1 - 126.4 - 2 - 139.7 - 3 7.57 d (2.5) 131.5 7.56 d (2.0) 4 - 136.2 5 7.40 dd (2.0; 8.5) 133.3 7.39 dd (2.0; 8.5) 6 8.11 d (8.5) 127.3 8.11 d (8.5) 7 - 161.7 1' - 145.5 2' - 127.9 3' 7.50 d (2.5) 130.3 7.50 d (2.5) 4' - 132.4 5' 7.32 dd (2.5; 8.5) 124.6 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 C G212-3 G212-3 TH δHa,b mult. (J, Hz) δCa,c δH a,b,# mult. (J, Hz) 1 - 170.9 3 5.13 s 66.7 5.13 s 3a - 134.7 4 - 137.1 5 - 150.6 6 6.72 s 118.4 6.72 s 7 - 130.1 7a - 113.5 8 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 cross- peaks 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 5 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% 2 steps; (d) LiAlH4, THF, 77%; (e) H2, Pd/C, THF, 97%; (f) BBr3, CH2Cl2, 0 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); 2 methoxy groups (C 57.1; 62.5, H 3.93; 4.11), 2 methylene groups and 5 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 (G212- 2′): 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 5 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, 2 methoxy groups and 5 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 G212- 5′ also indicated the correlations between CH3-10 protons with C-3a and C- 4. 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 Table 4.5. NMR data of G212-4′ and G212-5′ C G212-4′ Syn. from G212-3′ G212-4′ Syn. from G212-1′ G212-5′ δHa,b mult. (J, Hz) δCa,c δH a,b mult. (J, Hz) δHa,b mult. (J, Hz) δCa,c 1 - 169.0 - - 170.8 3 5.12 s 68.0 5.12 s 5.24 66.7 3a - 137.8 - - 138.8 4 - 120.6 - - 140.7 5 7.02 s 117.6 7.02 s - 156.0 6 - 152.5 - 6.81 s 116.1 7 - 146.5 - - 136.0 7a - 126.8 - - 115.3 8 4.05 s 62.3 4.05 s 3.90 s 56.3 9 3.90 s 57.0 3.90 s 3.95 s 60.4 10 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-7- methyl phthalide. G212-3 and its synthetic isomer (G212-6′) were both new compounds. 17 Table 4.6. NMR data of G212-3 TH and G212-6′ C G212-3 TH G212-6′ δHa,b mult. (J, Hz) δCa,c δHa,b mult. (J, Hz) δCa,c 1 170.9 169.8 3 5.13 s 66.7 5.11 s 68.1 3a 134.7 136.4 4 137.1 123.1 5 150.6 6.92 s 121.8 6 6.72 s 113.5 142.7 7 118.4 145.3 7a 111.5 111.5 8 2.38 16.0 2.08 s 16.4 OH-6 - 9.32 s - OH-7 - 9.67 s - 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 6 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), 1 coumarin compound: scopoletin (G278-8), 1 nucleoside: compound adenosine (G278-11), 1 dioxan compound: 2-((-5-methyl-1,4-dioxan-2-yl)methoxy)ethanol (G278- 12) and other phenolic compounds: benzyl salicylate (G278-9), N- phenylnaphthalen-2-amine (G278-10), N-(4-hydroxyphenylethyl)acetamide (G278-13), 2,4-dichlorophenyl 2,4-dichlorobenzoate (G278-14), 2,5-bis(5- tert-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, 3 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 G278- 15. 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 G278- 15 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 C G278-15 δHa,b mult. (J, Hz) δCa,c 2 - 157.5 3a - 141.3 4 7.72 d (8.0) 110.3 5 7.54 dd (2.0; 8.0) 123.9 6 - 148.3 7 7.81 d (2.0) 116.3 7a - 148.6 8 - 34.8 9 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, H- 5), 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 C G278-16 3-hydroxy-2-methylpyridine δHa,c mlult. (J, Hz) δCa,d δH b,e# mlult. (J, Hz) δC b,f# 2 - 147.7 - 144.7 3 - 154.0 - 146.3 4 7.09 d (8.0) 122.6 7.15 dd (1.2; 8.4) 122.3 5 7.01 dd (5.0; 8.0) 123.2 7.06 dd (4.7; 8.4) 122.4 6 7.86 d (5.0) 139.3 8.05 dd (1.2; 4.7) 139.7 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 3 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) N Comp. (*) Minimum Inhibitory Concentration - MIC (µg/mL) Gram positive Gram negative fungus E. faecalis ATCC2

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