Anti-MRSA actinomycins D1-D4 from the marine sponge-associated Streptomyces sp. LHW52447

Article abstract of DOI:10.1016/j.tet.2018.08.023

Actinomycins D₁?D₄ (1–4), four new D-type actinomycin analogues, were isolated from the fermentation broth of a strain of marine sponge-associated Streptomyces sp. LHW52447, together with actinomycin D (5). The structures of 1?4 were

Full text of DOI:10.1016/j.tet.2018.08.023

Accepted Manuscript
Anti-MRSA actinomycins D -D from the marine sponge-associated Streptomyces
1
4
sp. LHW52447
Wei-Hua Jiao, Wei Yuan, Zhi-Yong Li, Jing Li, Lei Li, Jia-Bao Sun, Yu-Han Gui, Jie
Wang, Bo-Ping Ye, Hou-Wen Lin
PII:
S0040-4020(18)30987-6
10.1016/j.tet.2018.08.023
TET 29743
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 10 July 2018
Revised Date: 13 August 2018
Accepted Date: 17 August 2018
Please cite this article as: Jiao W-H, Yuan W, Li Z-Y, Li J, Li L, Sun J-B, Gui Y-H, Wang J, Ye B-P, Lin
H-W, Anti-MRSA actinomycins D -D from the marine sponge-associated Streptomyces sp. LHW52447,
1
4
Tetrahedron (2018), doi: 10.1016/j.tet.2018.08.023.
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ACCEPTED MANUSCRIPT
Anti-MRSA Actinomycins D -D from the Marine Sponge-associated
1
4
Streptomyces sp. LHW52447
†,⊥
†,‡,⊥
§
†
†
†
Wei-Hua Jiao, Wei Yuan,
Zhi-Yong Li, Jing Li, Lei Li, Jia-Bao Sun, Yu-Han
†
†
,‡
,†
Gui, Jie Wang, Bo-Ping Ye,* and Hou-Wen Lin*
†
Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related
Genes, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao
Tong University, Shanghai 200127, People’s Republic of China
‡
Institute of life Science and Technology, China Pharmaceutical University, Nanjing
2
10009, People’s Republic of China
§
Key Laboratory of Microbial Metabolism, Marine Biotechnology Laboratory, School
of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai
2
00240, People’s Republic of China
1

ACCEPTED MANUSCRIPT
ABSRACT: Actinomycins D −D (1−4), four new D-type actinomycin analogues,
1
4
were isolated from the fermentation broth of a strain of marine sponge-associated
Streptomyces sp. LHW52447, together with actinomycin D (5). The structures of 1−4
were determined by a combination analysis of HRMS and NMR spectroscopic
methods, and their absolute configurations of amino acids were determined by
Marfey’s analysis. Actinomycins D (1) and D (2) are the first two naturally
1
2
occurring actinomycins with incorporation an oxazole unit into the central
phenoxazinone chromophore. Both 1 and 2 showed more potent antibacterial
activities against three strains of pathogenic methicillin-resistant Staphylococcus
aureus (MRSA) with MIC values of 0.125−0.25 µg/mL than those of 3−5 with MIC
values of 0.5−1.0 µg/mL, which suggested that the anti-MRSA activity might be
enhanced by the incorporation of an additional oxazole unit. In addition, the
cytotoxicity evaluation against WI-38 human diploid lung fibroblasts revealed that the
incorporation of oxazole unit could decrease the cytotoxicity of actinomycins on
human normal cells.
Keywords: Actinomycins; Marine Sponge; Streptomyces sp.; Structural elucidation;
Anti-MRSA activity.
2

ACCEPTED MANUSCRIPT
1
. Introduction
Actinomycins are a well-known class of chromopeptide isolated from many
1
species of Streptomyces. These metabolites consist of two cyclic pentapeptide
lactones (
α
- and
β
-rings) attached to a phenoxazinone chromophore through amide
2
bonds. About 34 naturally occurring actinomycins have been reported so far, while
more than 40 structural analogues have been obtained from precursor-directed
3
biosynthesis and synthetic efforts. This natural product class shows very potent
2
,4,5
antitumor and antibacterial activities and remains the subject of ongoing research.
6
These chromopeptides act as intercalators with the DNA double helix. The
phenoxazinone chromophore fits between guanine/cytosine base pairs, while the
7
peptidolactone side chains lying inside the minor groove of the helix. With some
notable exceptions, the structure elucidation of several actinomycins relies on mass
spectroscopic analyses, with absolute amino acid configurations often going
8
,9
unassigned. Such incomplete assignments could make it difficult to determine the
structure-activity relationship between structural analogues.
In an ongoing investigation of the chemical diversity of marine sponge-associated
1
0
microbes, our laboratory has accumulated an extensive library of microbial isolates
assembled from the marine sponges collected from the South China Sea. A strain of
Streptomyces sp. LHW52447, isolated from the marine sponge Phyllospongia
foliascens, was cultivated on a range of media, and metabolite production was
profiled by HPLC-DAD-MS analysis. The analysis revealed an array of interesting
metabolites (m/z >1200) in an organic extract derived from ISP2 broth medium.
3

ACCEPTED MANUSCRIPT
Scaled up cultivation followed by solvent extraction and partitioning, and reversed
phase chromatography, yielded a series of chromopeptides, actinomycins D −D (1−4)
1
4
and actinomycin D (5) (Figure 1). In structural characterization of 1−4 we
demonstrated a workflow capable of complete structure assignments, including
absolute configurations for all amino acid residues.
Figure 1. Actinomycins D −D (1−4) and actinomycin D (5) from the
1
4
sponge-associated Streptomyces sp. LHW52447.
2
. Results and discussion
Compound 1 was isolated as orange amorphous powders. The HRESI(+)MS
analysis of 1 showed a sodium adduct ion consistent with a molecular formula of
1
13
C H N O requiring 27 double-bond equivalents (DBE). The H and C NMR
63
86 12 16
data for 1 (Tables 1 and 2) displayed typical features of an actinomycin compound.
1
13
Detailed analysis of the H, C, COSY, HSQC, and HMBC NMR spectra allowed for
the assignment of the amino acids within the - and -rings as Thr, Val, Pro, Sar, and
MeVal (Figure 2). The amino acid composition of both rings is identical with
α
β
11
actinomycin D (5). However, the 12 amu difference between 1 and actinomycin D
indicated adduct of a carbon atom in 1, which was supported by an additional
4

ACCEPTED MANUSCRIPT
aromatic methine (δC 151.7/δH 8.19, CH-15). This methine proton showed HMBC
correlations with C-2 and C-3, suggesting the phenoxazinone chromophore in
actinomycin D was replaced by oxazolophenoxazine in 1. This assignment was
supported by HMBC correlations of 10-NH/C-1, C-4a, C-5a, and C-9 as well as
H -12/C-3, C-4, and C-4a. Furthermore, the amino acid sequences of
3
α
-ring and
β
-ring in 1 were determined by the NOESY correlations depicted in Figure 2, which
was confirmed by the TOF-MS/MS fragmentation (Figure S23). The absolute
configurations of amino acid residues in 1 was analyzed by 6 M HCl, followed by
1
0
Marfey’s analysis as described previously. The presence of
and -MeVal was unambiguously determined by comparison with authentic standards
Figure S24). Thus, the structure of 1 was determined as a new member of the
L-Thr, D-Val, L-Pro, Sar,
L
(
actinomycin family and subsequently named actinomycin D1.
5

ACCEPTED MANUSCRIPT
Figure 2. (A) Intra-residual COSY and HMBC connectivities for actinomycin D (1);
1
(B) inter-residual couplings for 1.
6

ACCEPTED MANUSCRIPT
Figure 3. Key NOESY Correlations for 1.
The molecular formula of 2 was determined to be C H N O on the basis of
67
94 12 16
HRESI(-)MS data. The NMR spectroscopic data for 2 (Tables 1 and 2) revealed
characteristic features of actinomycins and were quite similar to those of actinomycin
D (1), except for the resonances for an iso-butyl group (δC
1
/
δH 36.5/2.81, 27.1/2.23,
1
13
2
2.2/1.03, and 22.1/1.01) in the high field of H and C NMR spectra, supported by
the COSY correlations of H -16/H-17, H-17/H -18, and H-17/H -19. Moreover, the
2
3
3
placement of the iso-butyl group at C-15 ( C
δ
165.8) was determined by HMBC
correlations of H -16/C-15, C-18, and C-19 as well as H-17/C-15, C-16, and C-18.
2
The sequence and absolute configurations of amino acids in 2 were established by
TOF-MS/MS fragmentation (Figure S32) and Marfey’s analysis (Figure S33). The
overall structure of 2 was assigned as a new analogue of the actinomycin series and
designated as actinomycin D2.
7

ACCEPTED MANUSCRIPT
13
Table 1. C NMR Data (150 MHz) of 1−4
pentapeptidolaction (α-ring, δC
)
pentapeptidolaction (β-ring, δC)
a
b
a
a
a
b
a
a
no.
1
2
3
4
no.
1
2
3
4
1
1
L-Thr
L-Thr
1
2
3
4
168.7, C
55.0, CH
70.5, CH
17.1, CH3
169.2, C
53.9, CH
72.3, CH
16.4, CH3
168.8, C
53.8, CH
73.8, CH
16.9, CH3
168.6, C
55.2, CH
75.0, CH
17.9, CH3
1
169.2, C
56.6, CH
71.0, CH
17.5, CH3
169.3, C
54.7, CH
72.0, CH
16.7, CH3
168.4, C
54.2, CH
73.4, CH
16.7, CH3
168.9, C
54.9, C
2
3
75.4, CH
17.5, CH3
4
2
2
2
D-Val /D-Ala
D-Val
1
2
3
4
5
171.3, C
56.2, CH
29.7, CH
19.2, CH3
18.1, CH3
170.6, C
57.4, CH
30.2, CH
18.7, CH3
19.1, CH3
173.6, C
58.7, CH
31.7, CH
18.7, CH3
19.3, CH3
173.5, C
48.8, CH
17.7, CH3
1
2
3
4
5
170.4, C
56.1, CH
30.2, CH
19.3, CH3
18.1, CH3
170.6, C
57.4, CH
30.2, CH
18.7, CH3
19.1, CH3
173.6, C
58.7, CH
31.7, CH
18.7, CH3
19.2, CH3
173.8, C
58.8, CH
32.0, CH
19.3, CH3
19.1, CH3
3
3
L-Pro
L-Pro
1
2
3
4
5
172.9, C
173.0, C
173.3, C
173.4, C
1
2
3
4
5
172.6, C
173.0, C
173.1, C
173.6, C
55.4, CH
28.5, CH2
24.7, CH2
46.8, CH2
54.7, CH
31.3, CH2
22.8, CH2
46.4, CH2
55.8, CH
31.8, CH2
23.3, CH2
47.2, CH2
56.4, CH
31.4, CH2
23.1, CH2
47.7, CH2
55.6, CH
28.1, CH2
24.8, CH2
47.0, CH2
54.8, CH
31.2, CH2
22.7, CH2
46.4, CH2
55.8, CH
31.8, CH2
23.3, CH2
47.2, CH2
56.6, CH
31.1, CH2
23.3, CH2
47.5, CH2
4
4
Sar
Sar
1
170.5, C
167.2, C
166.5, C
166.6, C
1
169.6, C
167.0, C
166.3, C
166.7, C
8

ACCEPTED MANUSCRIPT
2
49.6, CH2
38.7, CH3
51.2, CH2
34.4, CH3
51.7, CH2
34.9, CH3
51.5, CH2
35.1, CH3
2
50.0, CH2
38.0, CH3
51.1, CH2
34.4, CH3
51.5, CH2
34.9, CH3
51.6, CH2
35.3, CH3
NMe
NMe
5
5
L-MeVal
L-MeVal
1
168.9, C
168.2, C
167.4, C
167.8, C
71.2, CH
27.1, CH
21.7, CH3
19.2, CH3
39.3, CH3
1
169.2, C
60.5, CH
28.0, CH
19.1, CH3
18.6, CH3
30.5, CH3
168.1, C
167.5, C
167.7, C
71.4, CH
27.3, CH
21.7, CH3
19.2, CH3
39.4, CH3
2
60.4, CH
27.8, CH
18.8, CH3
18.6, CH3
30.5, CH3
69.9, CH2
26.3, CH
21.1, CH3
18.8, CH3
38.6, CH3
71.6, CH
26.6, CH
21.7, CH3
19.1, CH3
39.4, CH3
2
69.5, CH
26.5, CH
21.1, CH3
18.8, CH3
38.7, CH3
71.5, CH
26.8, CH
21.6, CH3
19.1, CH3
39.4, CH3
3
3
4
4
5
5
NMe
NMe
chromophore
1
2
3
4
101.0, C
132.4, C
143.8, C
112.1, C
141.3, C
141.3, C
127.9, C
122.3, CH
122.3, CH
112.9, C
100.2, C
132.1, C
143.5, C
111.8, C
139.5, C
140.8, C
127.5, C
122.1, CH
122.1, CH
113.6, C
131.3, C
133.1, C
132.5, C
180.7, C
114.4, C
145.1, C
141.8, C
126.9, C
133.6, C
123.4, C
136.1, C
129.6, C
102.0, C
147.6, C
179.3, C
113.6, C
145.2, C
140.6, C
127.8, C
130.4, CH
126.0, CH
132.7, C
129.3, C
10a
11
12
13
14
15
16
17
18
19
133.3, C
15.2, CH3
9.0, CH3
168.2, C
166.8, C
151.7, CH
133.1, C
14.9, CH3
9.0, CH3
166.5, C
165.4, C
165.8, C
36.5, CH
27.1, CH
22.2, CH3
22.1, CH3
146.9, C
14.9, CH3
7.8, C
146.0, C
15.2, CH3
7.9, CH3
166.3, C
166.7, C
166.7, C
162.6, C
169.9, C
24.1, CH3
4
5
6
7
8
9
a
a
9
a
132.5, C
a
b
Measrued in CDCl . Measured in DMSO-d .
3
6
9

ACCEPTED MANUSCRIPT
1
Table 2. H NMR Data (600 MHz) of 1−4
pentapeptidolaction (α-ring, δC
)
pentapeptidolaction (β-ring, δC)
a
b
a
a
a
b
a
a
no.
L-Thr
2
1
2
3
4
no.
L-Thr
2
1
2
3
4
1
1
4.96, dd (9.2, 4.87, dd (8.9, 4.47, dd (6.7,
.4) 1.6) 2.6)
5.80, qd (6.6, 5.14, qd (6.1, 5.43, qd (6.2, 5.17, m
4.89, dd (8.7, 4.76, d (9.7)
4.58, dd (6.2,
2.6)
5
.18, d (9.6)
4.91, m
2
2.3)
3
3
5.23, qd (6.2, 5.36, q (6.3)
1.7)
5.17, m
5
.66, q (6.4)
2
.3)
1.9)
1.5)
4
1.19, d (6.4)
1.16, d (6.4)
1.36, d (6.2)
6.72, d (8.9)
1.24, m
7.27, m
4
1.29, d (6.4) 1.18, d (6.3)
1.26, d (6.3)
7.38, m
1.24, m
NH
NH
10.01,
8.5)
d
7.74, d (6.8)
8.97, d (9.4)
7.27, d (9.0)
9.72, d (8.9)
(
2
2
D-Val /
D-Val
2
D-Ala
2
4.36, m
3.46, m
1.87, m
0.92, m
3.64, m
3.93, m
2
4.49, m
1.90, m
0.91, m
0.84, m
3.47, m
3.66, m
3.56, m
3
1.91, m
2.14, m
1.35, d (6.7)
8.09, d (5.9)
3
1.88, m
2.16, m
2.19, m
4
0.91, m
1.14, d (6.2)
0.89, d (6.4)
8.67, d (6.0)
4
0.93, m
1.14, d (6.2)
0.89, d (6.4)
8.58, d (6.2)
0.92, d (6.8)
1.13, d (6.7)
8.21, d (6.3)
o
5
0.81, m
0.69, d (6.7)
5
0.69, d (6.7)
NH
6.43, d (8.4)
8.40, d (5.8)
NH
7.13, d (8.5) 8.33, d (5.8)
3
3
L-Pro
L-Pro
2
2
6.33, dd (9.6, 5.97, d (9.2)
.0)
2.38, m
6.24, dd (8.8, 5.76, d (9.0)
2.4)
4
.89, m
6.26, d (8.4)
2.08, m
4.87, m
2.05, m
6.20, d (7.9)
2.10, m
2
3
a
2.05, m
2.93, m
3
2.32, m
2.59, m
1
0

ACCEPTED MANUSCRIPT
3
4
4
5
5
b
a
b
a
b
1.92, m
2.30, m
1.94, m
3.91, m
3.56, m
1.74, m
1.91, m
1.67, m
3.47, m
3.26, m
1.86, m
2.15, m
1.98, m
3.88, m
3.64, m
1.86, m
1.92, m
2.36, m
1.94, m
4.11, m
3.52, m
1.74, m
1.90, m
1.67, m
3.49, m
3.25, m
1.86, m
2.15, m
1.93, m
3.84, m
3.62, m
1.89, m
2.20, m
2.09, m
3.91, m
3.73, m
2.27, m
2.09, m
3.83, m
3.73, m
4
5
4
4
Sar
Sar
2
2
2
a
4.77, m
3.05, m
3.42, s
4.77, m
4.93, d (17.3)
3.67, m
4.78, d (16.8)
3.61, d (17.5)
2.88, s
4.91, m
3.07, m
3.31, s
4.74, m
4.81, d (17.4)
3.66, m
4.76, d (17.0)
3.68, d (17.6)
2.88, s
b
4.12, d (18.0)
2.75, s
4.08, d (18.0)
2.75, s
NMe
L-MeVal
2
2.90, s
NMe
L-MeVal
2
2.90, s
5
5
2.82, d (9.9)
2.68, d (8.8)
5.03,
d
2.80, d (9.8)
2.69, d (9.0)
4.74, m
3.13, d (9.0)
3.22, m
(11.0)
3
2.17, m
0.84, m
0.78, m
3.21, s
2.51, m
2.74, m
2.60, m
3
2.21, m
0.92, m
0.82, m
3.09, s
2.56, m
2.74, m
2.66, m
4
0.98, d (6.6)
0.79, d (7.2)
3.19, s
1.01, d (5.9)
0.81, d (6.8)
3.18, s
0.96, d (6.6)
0.75, d (6.6)
2.93, s
4
0.97, d (6.6)
0.81, d (7.2)
3.22, s
1.01, d (5.9)
0.79, d (6.7)
3.02, s
0.96, d (6.6)
074, d (6.6)
2.90, s
5
5
NMe
NMe
chromophore
7
8
6.58, d (7.9)
6.64, d (8.3)
7.13, d (8.1)
2.16, s
7.39, d (8.4)
7.27, d (8.4)
2.48, s
7.36, d (7.9)
7.65, d (7.5)
2.55, s
16
17
18
19
NH
2.81, m
2.16, s
7.60, d (8.2)
2.15, s
2.23, m
11
1.03, d (6.7)
1.01, d (6.6)
11.77, s
1
2
5
2.32, s
2.30, s
2.15, s
2.24, s
1
8.19, s
11.97, s
a
b
Measrued in CDCl . Measured in DMSO-d .
3
6
1
1

ACCEPTED MANUSCRIPT
Compound 3 was assigned the molecular formula C H N O by the
6
4
88 12 17
HRESI(-)MS data. The NMR data showed the presence of the phenoxazinone
chromophore and two identical pentapeptidolactones that contained Thr, Val, Pro, Sar,
and MeVal moieties (Tables 1 and 2). Compound 3 differed with actinomycin D by an
additional acetylation of 2-NH (δC 169.9, C-15, and δC 24.1/δH 2.16, CH -16), which
3
was supported by the 2D NMR analysis. In combination with the TOF-MS/MS
fragmentation (Figure S41) and Marfey’s analysis (Figure S42), the data were
consistent with structure of 3 as depicted and named actinomycin D3.
The molecular formula of compound 4 was determined as C H N O by the
6
0
82 12 16
HRESI(-)MS data with less of 28 amu (C H ) than actinomycin D. The NMR data
2
4
recorded for 4 was nearly identical with those of actinomycin D, except for the
replacement of Val by Ala in -ring of 2 (Tables 1 and 2). Further COSY and HMBC
correlations were full agreement with Ala in -ring. Marfey’s anlysis revealed the
α
α
D-configuration of Ala (Figure S51). Thus the whole structure of 4 was assigned as
shown and named actinomycin D4.
The antibacterial activity of the isolated actinomycins was evaluated against three
1
2
strains of pathogenic methicillin-resistant Staphylococcus aureus (MRSA), using
chloramphenicol and daptomycin as positive control (Table 3), the anti-MRSA
activities of 1 and 2 are nearly 2−4 times potent than those of 3−5, which suggested
the anti-MRSA activity might be enhanced by incorporation of an additional oxazole
unit into the phenoxazinone chromophore. In addition, the cytotoxic activity of the
five isolates was evaluated against WI-38 human diploid lung fibroblasts (Table 3).
The results showed that 1 and 2 showed less cytotoxicity against human normal WI38
cells than 3−5, which suggested that the incorporation of oxazole unit into the
chromophore could more dramatically decrease the cytotoxicity of actinomycins
1
2

ACCEPTED MANUSCRIPT
against human normal cells.
Table 3. Anti-MRSA activity and cytotoxicity of 1−5 against three strains of pathogenic
methicillin-resistant Staphylococcus aureus (MRSA) and WI-38 human diploid lung fibroblasts.
MIC (µg/mL)
P175
IC (µM)
50
Compound
P172
0.125
0.25
0.5
ATCC 33591
0.125
0.25
WI38
74 ± 10
126 ± 6
18 ± 2
23 ± 4
11 ± 3
1
0.25
0.25
1.0
2
3
0.5
4
0.5
0.25
0.25
0.5
0.25
5
0.5
0.25
a
chloramphenicol
0.5
1.0
a
daptomycin
0.063
0.031
0.25
a
Adriamycin
62 ± 22
a
positive control
3
. Conclusion
In summary, four new D-type actinomycin analogues, actinomycins D −D (1−4),
1
4
were isolated from the fermentation broth of a strain of marine sponge-associated
Streptomyces sp. LHW52447. Antibacterial and cytotoxicity assays showed that the
incorporation of oxazole unit into the phenoxazinone chromophore would enhance the
antibacterial activity of actinomycins while decrease their cytotoxicity against human
normal cells. This represents the first actinomycin compounds from a marine source
and highlights the importance of continued efforts toward screening for chemical
diversity within sponge-associated microbes.
4
. Experimental Section
4
.1 General Experimental Procedures
Specific optical rotations ([
α]D) were measured on an Autopol I polarimeter (No.
3
0575, Rudolph Research Analytical) with a 10 cm length cell at room temperature.
NMR spectra were recorded at room temperature on a Bruker Avance DRX-600 MHz
NMR spectrometer with DMSO-d and CDCl as the solvent and internal standard.
6
3
1
3

ACCEPTED MANUSCRIPT
Spectra were referenced to residual solvent signals with resonances at δ 2.49/δ 39.5
H
C
for DMSO-d and δ 7.26/δ 77.0 for CDCl . Column chromatography was conducted
6
H
C
3
using silica gel (65 × 250 or 230 × 400 mesh). Sephadex LH-20 was purchased from
Amersham Pharmacia Biotech AB, Uppsala, Sweden. Analytical thin-layer
chromatography (TLC) was performed on silica gel 60 F254 plates. Purification of the
compounds was performed using a Waters Alliance 2695 separation module equipped
with a Waters 2998 photodiode array (PDA) detector.
4
.2 Collection and Isolation of Streptomyces sp. LHW52447
The Streptomyces sp. LHW52447 was isolated in 2013 from the specimen of
marine sponge Phyllospongia foliascens, collected from the Xisha Islands in the
South China Sea. The marine sponge sample was cut into pieces and dissected under
aseptic conditions and placed on ISP2 agar plates (comprising 0.4% glucose, 0.4%
yeast extract, 1% malt extract, 0.2% CaCO , 0.02% chloramphenicol, and 1.5% agar
3
in 50 % sterile water). Agar plates were sealed and incubated at 26.5 °C for 3-4 weeks.
A pure culture of bacterial LHW52447 was obtained by single-colony serial transfer
on agar plates. Taxonomic analysis identified LHW52447 as Streptomyces sp.
4
.3 Fermentation and Fractionation
The strain of Streptomyces sp. LHW52447 was cultivated in ten 1 L Erlenmeyer
flasks, each containing 250 mL ISP2 medium. After 3 days of incubation at 28 °C
with 200 rpm agitation, the cultures were used to incolate ten flasks (1 L), each
containing 250 mL of ISP2 medium. The fermentation was continued for 7 days at 28
°C with 200 rpm agitation. All culture flasks were combined and extracted with
EtOAc until the organic phase was colorless. The combined organic phase was
concentrated in vacuo to yield a crude extract (9.6 g), which was subjected to VLC on
silica gel using gradient of n-hexane/acetone (from 100:1 to 0:1) to obtain eight
1
4

ACCEPTED MANUSCRIPT
fractions (A−H). HPLC-DAD-MS analysis revealed that actinomycin compounds
were mainly distributed in fractions F and G. Both of the two fractions were further
separated by reversed-phase MPLC (gradient MeOH/H O, from 10% to 100% for 180
2
min, flow rate 20 mL/min, UV detection at 210 nm), and the subfractions were further
purified by semi-preparative reversed-phase HPLC (YMC C RS column, 250 × 10
18
mm, 5 µm, 2 mL/min) to afford actinomycin D (3) (45% MeCN/H O, t = 24.1 min,
3
2
R
7
.7 mg), actinomycin D (4) (50% MeCN/H O, t = 60.0 min, 8.3 mg), actinomycin D
4 2 R
(
5) (55% MeCN/H O, t = 20.2 min, 47.5 mg), actinomycin D (1) (55% MeCN/H O,
2 R 1 2
t = 27.3 min, 8.9 mg), and actinomycin D (2) (60% MeCN/H O, t = 60.1 min, 3.8
R
2
2
R
mg).
4
.3.1 Actinomycin D (1)
1
Orange solids; [α] -116.9 (c 0.1, MeOH)；UV (MeOH) (log ε) λ 225 (4.10),
D
max
2
52 (4.06), 409 (3.71) nm; IR (KBr) νmax 3418, 3269, 2964, 2933 2875, 1744, 1651,
-1
1
13
1
504, 1454, 1259, 1192, 734 cm ; H and C NMR data, Tables 1 and 2;
+
HRESI(+)MS m/z 1289.6185 [M + Na] (calcd for C H N O Na, 1289.6182).
63
86 12 16
4
.3.2 Actinomycin D (2)
2
Orange solids; [α] -85.5 (c 0.15, MeOH)；UV (MeOH) (log ε) λ 228 (4.13),
D
max
2
1
52 (4.12), 410 (3.79) nm; IR (KBr) νmax 3445, 3268, 2966, 2933, 2876, 1747, 1652,
-1
1
13
504, 1449, 1256, 1192, 1093 cm ; H and C NMR data, Tables 1 and 2;
-
HRESI(-)MS m/z 1321.6829 [M - H] (calcd for C H N O , 1321.6833).
6
7
93 12 16
4
.3.2 Actinomycin D (3)
3
Orange solids; [α] -51.4 (c 0.15, MeOH)；UV (MeOH) (log ε) λmax 214 (3.97),
D
3
76 (3.33), 460 (3.05) nm; IR (KBr) νmax 3400, 3254, 2962, 2997 2855, 1747, 1669,
-1
1
13
1
622, 1513 1470, 1299, 1192, 822, 737 cm ; H and C NMR data, Tables 1 and 2;
-
HRESI(-)MS m/z 1295.6305 [M - H] (calcd for C H N O , 1295.6312).
6
4
87 12 17
1
5

ACCEPTED MANUSCRIPT
4
.3.3 Actinomycin D (4)
4
Orange solids; [α] -89.4 (c 0.15, MeOH)；UV (MeOH) (log ε) λmax 214 (3.97), 376
D
(3.33), 460 (3.05) nm; IR (KBr) νmax 3445, 3268, 2966, 2933, 2876, 1747, 1652, 1504,
-
1
1
13
1
1
4
449, 1256, 1192, 1093 cm ; H and C NMR data, Tables 1 and 2; HRESI(-)MS m/z
-
225.5887 [M - H] (calcd for C H N O , 1225.5894).
6
0
81 12 16
.4 Acid Hydrolysis of 1−4 and Absolute Configurations of Amino Acids by Marfey’s
analysis
1
0,13
Analysis was carried out following the published method.
compounds 1 4 (0.1 mg) in 6 M HCl (100 L) were heated to 110 C in a sealed vial
for 12 h, and then the hydrolysates were concentrated under a stream of dry N . The
Samples of
−
µ
°
2
hydrolysates were then treated with 1 M NaHCO3 (40
1-fluoro-2-4-dinitrophenyl-5-L-leucine amide) (1% solution in acetone, 50
h, after which the reaction was neutralized with 1M HCl. Analytes and the authentic
amino acid standards prepared from treatment of -Thr, -Val, -Pro, -MeVal, and
-Ala with both -FDAA and -FDAA, were subjected to HPLC-ESI -MS analysis.
The retention times of the - and -/ -FDLA-derivatized amino acids were measured
by UPLC-MS using an Acquity UPLC BEH C column (2.1 × 50 mm, 1.7 ꢀm, 0.5
µ
L) and
L-FDLA
(
µL) for 2
L
D
L
L
D
D
L
L
L D
1
8
mL/min) with a linear gradient from 10% to 100% MeCN/H O containing 0.1%
2
formic acid over 10 min. The absolute configurations of amino acids were established
by comparison of the retention times of the
L- and D-FDLA derivatives of
corresponding amino acids. The retention times of the
L-FDLA amino acid derivatives
+
+
were 3.07 min (
L
-Thr, m/z 414 [M + H] ), 4.82 min (
D
-Val, m/z 412 [M + H] ), 3.59
+
+
min (
L
-Pro, m/z 410 [M + H] ), 4.36 min (
L
-MeVal, m/z 426 [M + H] ), and 4.06 min
+
(D
-Ala, m/z 384 [M +H] ).
4
.5 Cytotoxicity Assay
1
6

ACCEPTED MANUSCRIPT
Cytotoxicity of the five isolates were evaluated against human lung WI38
1
4
embryonal firoblasts using a reported protocol after slight modifications. Cells were
cultured in 96-well plates in 80 ꢀL of medium in a humidified 37 C incubator
°
supplied with 5% CO . The test compounds were added in a 25 ꢀL aliquot generated
2
by serial dilution in serum-free medium on the day of the experiment, after prior
removal of 25 ꢀL of media from the treated well. Aliquots were generated from stock
solutions of 6 mg/mL compound in 100% DMSO. The test compounds were
evaluated in WI38 cells for 48 h treatment. Each compound was tested at final
concentrations ranging from 0.1 to 30 ꢀM. In all cases, cell viability was determined
after 48 h treatment using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay. The cytotoxicity of each test compound was
assessed in at least three independent cultures. The IC50 values were derived by
nonlinear regression analysis.
4
.6 Antibacterial Assay
Minimum inhibitory concentration (MIC) was determined according to Clinical
1
1
and Laboratory Standards Institute (CLSI) guidelines. The MIC50 values were
recorded using a spectrophotometer. For antibiotic sensitivity assays, bacteria in
9
6-well plates (Corning) were incubated with 1−5 or antibiotic standards at final
concentrations of 0 to 64 µg/mL. The plates were incubated at 37 °C and read at 24 h.
Author information
Corresponding Author
*
E-Mail: yebp@cpu.edu.cn (B. P. Ye);
Tel: +86-21-68383346. Fax: +86-21-58732594. E-mail: franklin67@126.com (H. W.
*
Lin).
1
7

ACCEPTED MANUSCRIPT
ORCID
Wei-Hua Jiao: 0000-0003-4835-4775
Hou-Wen Lin: 0000-0002-7097-0876
Author Contributions
⊥
W. H. Jiao and W. Yuan contributed equally to this work.
Notes
The authors declare no competing financial interest.
Acknowledgments
This research was supported by the National Natural Science Fund of China (Nos.
8
1741149, 41476121, 41576130, 31600014, and U1605221), and the Fund of the
Science and Technology Commission of Shanghai Municipality (No. 14431901300).
Appendix A. Supplementary data
Copies of 1D and 2D NMR, HRESIMS, QTOF-MS/MS, and Marfey’s analysis of
1
−4. These materials are available free of charge via the Internet at http:…
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Graphical abstract
2
0