Nghiên cứu phát hiện các hợp chất thứ cấp có hoạt tính kháng vi sinh vật kiểm định phân lập từ một số chủng xạ khuẩn biển thuộc chi streptomyces tt tiếng anh

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.


1

INTRODUCTION
1. 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.
2. 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 2 promising
isolated actinobacteria strains.
- Determination the antimicrobial activities of isolated compounds.


2

3. Main research activities
1. Isolation and culture actinobacteria strains from collected marine
samples;
2. Screening the antimicrobial activities of isolated strains, select two
promising strains for identification and fermentation (50L);
3. Isolation of secondary metabolites from 2 fermented strains;
4. Structure elucidation of isolated compounds;

5. Determination of antimicrobial activities of isolated compounds.
CHAPTER 1. 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 2. 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)


3

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 3. 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, 3 algae, 3 soft
corals and 1 sponge).
- 14 samples from Cu Lao Cham, Quang Nam (5 sediments, 2 soft
corals, 1 sponge, 2 echinoderms, 2 sea-rabbits, 1 snake tail and 1 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 4 test microbial pathogens. Two strain (G212 and G278),
inhibited 5 tested pathogens, representing two marine areas, were selected
for further study.


4

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), 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 2,4dichlorobenzoate (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), 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), 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)


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)
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

1
2
3
4
5
6
7

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


7
8
9
1

2
3
4
5
6
7
8
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
8
G277
256
G278
256

32
16
16
2
G280
256
256
16
32
32
G283
128
G284
256
256
G285
32
G288
32
G289
32
G290
32
16
8
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


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 (G2122), 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-2carboxylic (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 (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


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.
Poly(ethylene
G212-1
terephthalate)
C
δHa,c mult. (J, Hz)
δCa,d
δC a,e,#
1
165.3
168
2

-

133.8

134

3
4

8,10 s
4,69 s

129.7
62.7

130
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

13

C-NMR data is consistent with a molecular formula of

1

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 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 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 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
G212-2
G212-2 TH
C
a,b
a,c

a,b,#
δH mult. (J, Hz)
δC
δH
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
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)
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 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 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 (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 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 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

1
3
3a
4
5
6
7
7a
8
9
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
1
3
3a
4

5
6
7
7a
8
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 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 (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, 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 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
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, 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#
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)
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

1
2
3
4
5
6

7
8
9
10
11
12
13
14
15
17
18
19
20
1
2
3

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
1. 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.
4. 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.
5. Testing isolated compounds against a panel of microbial
pathogens revealed that 20/30 isolates shown inhibition activities against
tested pathogens.
6. 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
1. Further biological characterizations should be conducted for isolated
compounds that exhibited antimicrobial activities.
2. 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
1. 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 4 tested micro-organisms.
2. 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.

3. 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.
4. All obtained compounds were screened for their antimicrobial
activities. Results revealed that 20/30 isolates and 3 synthetic compounds shown
inhibition activities against tested microbial pathogens.