A new analogue of echinomycin and a new cyclic dipeptide from a marine-derived streptomyces sp. LS298

Article abstract of DOI:10.3390/md13116947

Quinomycin G (1), a new analogue of echinomycin, together with a new cyclic dipeptide, cyclo-(L-Pro-4-OH-L-Leu) (2), as well as three known antibiotic compounds tirandamycin A (3), tirandamycin B (4) and staurosporine (5), were isolated from Streptomyces

Full text of DOI:10.3390/md13116947

Mar. Drugs 2015, 13, 6947-6961; doi:10.3390/md13116947
OPEN ACCESS
marine drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
A New Analogue of Echinomycin and a New Cyclic Dipeptide
from a Marine-Derived Streptomyces sp. LS298
†
†
Xin Zhen , Ting Gong , Fu Liu, Pei-Cheng Zhang, Wan-Qi Zhou, Yan Li and Ping Zhu *
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
1
Xian Nong Tan Street, Xicheng District, Beijing 100050, China;
E-Mails: zhenxin@imm.ac.cn (X.Z.); gongting@imm.ac.cn (T.G.); liufu@imm.ac.cn (F.L.);
pczhang@imm.ac.cn (P.-C.Z.); zhouwanqi@imm.ac.cn (W.-Q.Z.); liyanxiao@imm.ac.cn (Y.L.)
†
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed; E-Mail: zhuping@imm.ac.cn;
Tel.: +86-10-6316-5197.
Academic Editor: Orazio Taglialatela-Scafati
Received: 2 July 2015 / Accepted: 6 November 2015 / Published: 18 November 2015
Abstract: Quinomycin G (1), a new analogue of echinomycin, together with a new cyclic
dipeptide, cyclo-(L-Pro-4-OH-L-Leu) (2), as well as three known antibiotic compounds
tirandamycin A (3), tirandamycin B (4) and staurosporine (5), were isolated from
Streptomyces sp. LS298 obtained from a marine sponge Gelliodes carnosa. The planar and
absolute configurations of compounds 1 and 2 were established by MS, NMR spectral data
analysis and Marfey’s method. Furthermore, the differences in NMR data of keto-enol
tautomers in tirandamycins were discussed for the first time. Antibacterial and anti-tumor
activities of compound 1 were measured against 15 drug-sensitive/resistant strains and
1
2 tumor cell lines. Compound 1 exhibited moderate antibacterial activities against
Staphylococcuse pidermidis, S. aureus, Enterococcus faecium, and E. faecalis with the
minimum inhibitory concentration (MIC) values ranged from 16 to 64 μg/mL. Moreover, it
displayed remarkable anti-tumor activities; the highest activity was observed against the
Jurkat cell line (human T-cell leukemia) with an IC50 value of 0.414 μM.
Keywords: marine-derived Streptomyces; secondary metabolites; antibacterial activity;
anti-tumor activity

Mar. Drugs 2015, 13
. Introduction
With the emergence of newer resistant forms of infectious diseases and multi-drug resistant (MDR)
6948
1
bacteria and tumors, it has become essential to develop novel and more effective antibiotics [1].
In recent years, numerous studies have discovered that marine-derived actinomycete strains, mainly
Streptomyces species, have the ability to produce a wide variety of biologically active and structurally
unique metabolites. Some of these compounds possess strong antibacterial and anti-tumor activities [2–4].
The immense diversity of marine actinomycetes, along with their underutilization, has attracted great
attention from researchers to discover novel antibiotics [5–8].
The strain LS298 was obtained from a marine sponge Gelliodes carnosa collected from the South
China Sea. Based on the 16S rRNA sequence (GenBank accession number FJ937945) analysis [9] and
the morphology, this strain was preliminarily identified as Streptomyces sp. Our previous studies have
shown that the secondary metabolites of this strain contain echinomycin, cyclic dipeptides, and esters [10].
Among these compounds, echinomycin, a bifunctional DNA intercalator, is the predominantly and
biologically active constituent against the Gram-positive and Gram-negative bacteria and also shows
good anti-tumor activity [11–14]. Our continued search for echinomycin analogues or other novel
antibiotics from extracts of large scale fermentation led to the isolation of two new compounds
quinomycin G (1) and cyclo-(L-Pro-4-OH-L-Leu) (2), as well as three known compounds tirandamycin
A (3), tirandamycin B (4), and staurosporine (5) (Figure 1). Structurally, quinomycin G (1) possessed a
terminal double bond in one of the Ser groups. Cyclo-(L-Pro-4-OH-L-Leu) (2) was a new cyclic
dipeptide. Tirandamycin A (3) was the 1-enol-4′-keto form, while tirandamycin B (4) was 1-keto-4′-enol
form. It is the first time to reveal this form of tirandamycin B explicitly. In addition, antibacterial and
anti-tumor activities of compound 1 were evaluated against 15 drug-resistant/sensitive strains and 12 tumor
cell lines.
5
5
Ser
Ala N-Me-Cys N-Me-Val
10
N4 3
2
N4 3
1
0
6
7
O
O
6
7
O
O
N
H
H
2
N
N
O
N
9
N
N
H
N
O
9
N
N
H
N
O
COOH
8
8
1
1
O
O
N
O
O
O
N
S
S
S
S
O
O
O
O
1'
1
'
H
N
H
8'
8
'
2'
3'
N
9'
N
2'
3'
N
9'
O
N
N
7'
6'
O
N
N
7'
H
H
O
O
O
O
6
'
N
4'
10'
N
10'
5'
5
'
4
'
N-Me-Val' N-Me-Cys' Ala' Dehydroxy-Ser
1
Echinomycin
1
8
O 11
1
8
O 11
H
10O
HO
10O
N
1
1
2
3
O
1
2
1
3
7
7
O
6
O
6
14
16
1
4
16
O
O
O
9
1
0
H
9
8
17
8
17
1
2
1
2
5
1
1
3
5
5
1
5
N
15
9
4
HO
6
4
4
N
N
HN
3
1
3
3
O
8
7
O
2
H
2
1
3' 2'
1
O
O
HO
3
'2'
O
3
H CO
1
'
4'
1'
NH
4
'
NHCH
NH
HO
3
O
5
'
5
'
2
3
4
5
Figure 1. Structures of the isolated compounds 1–5 and reference compound echinomycin.

Mar. Drugs 2015, 13
6949
2
. Results and Discussion
2
.1. Structure Elucidation of Compounds 1–5
Quinomycin G (1) was obtained as an amorphous yellow powder, a molecular formula of
+
C
51
H
64
N
12
O
12
S
2
was determined by HRESIMS (m/z 1101.4288 [M + H] , calcd for C51
H
65
N
12
O
12
2
S ,
1
101.4286), requiring 26 degrees of unsaturation. The chemical structure of 1 was adumbrated as an
echinomycin analogue by the close similarity of its molecular formula and ultraviolet spectral
properties (λmax (log ε) 245.2 nm (2.6), 325.8 nm (1.9), respectively) to those of echinomycin [10].
1
The H NMR spectrum of 1 (Table 1) displayed four NH resonances (δ
H
: 10.67 (1H, s), 9.20 (1H, d,
: 9.68 (1H, s),
.63 (1H, s), 8.27 (1H, d, J = 8.0 Hz), 8.20 (3H, d, J = 8.0 Hz), 7.95–7.97 (2H, overlap), 7.84–7.89
2H, overlap), 6.90 (1H, brs), 6.11 (1H, brs)); two methylene resonances (δ : 5.02 (1H, dd, J = 11.5,
.0 Hz), 4.67 (1H, d, J = 11.5 Hz); 3.47 (1H, dd, J = 16.0, 5.0 Hz), 2.54 (1H, d, J = 16.0 Hz));
: 6.03 (1H, d, J = 4.0 Hz), 5.70 (1H, s), 5.37 (1H, d, J = 9.0 Hz), 5.28 (1H, m),
.85 (1H, m), 4.47 (1H, d, J = 11.0 Hz), 3.74 (1H, d, J = 2.0 Hz), 3.42 (1H, d, J = 10.5 Hz), 2.49 (1H, m),
.27 (1H, m)); 11 methyl signals in the upfield region, including four N-Me groups (δ : 3.37 (3H, s),
.15 (3H, s), 3.02 (3H, s), 2.97 (3H, s)), one S-Me group (δ : 2.07 (3H, s)). 51 carbons were observed
J = 9.5 Hz), 9.01 (1H, d, J = 9.5 Hz), 7.83 (1H, overlap)); 12 aromatic protons signals (δ
H
9
(
H
3
ten methine signals (δ
H
4
2
3
H
H
1
3
in the C NMR spectrum of compound 1 (Table 1), including ten ester/amide carbonyls (δ
C
: 172.2
2
(
2C), 171.5, 169.9 (2C), 169.8, 168.3, 163.6, 163.2, 161.9) and 18 sp carbon signals (δ
C
: 143.9 (2C),
1
43.7, 143.5, 143.4, 142.4, 140.3 (2C), 133.0, 132.4, 131.9, 131.6, 131.0, 130.0 (2C), 129.4 (2C),
1
1
1
04.3). Comprehensive analysis of the H- H COSY (Supplementary Materials Figure S8) and HSQC
of compound 1, indicated that compound 1 was comprised of two quinoxalines and eight amino acid
moieties (two N-Me-Val, two Ala, two N-Me-Cys, one Ser, and one Dehydroxy-Ser) (Figure 2).
The connections between amino acids moieties were confirmed by an HMBC experiment.
The HMBCs ((H-α (δ
.37 (3H, s) of N-Me-Cys′ to the C=O (δ
C=O (δ : 171.5) of N-Me-Cys′; H-β (δ : 4.67 (1H, d, J = 11.5 Hz) of Ser to the C=O (δ
N-Me-Val′; NH (δ : 9.20 (1H, d, J = 9.5 Hz) of Ala to the C=O (δ : 169.9) of Ser; N-CH
3H, s) of N-Me-Cys to the C=O (δ : 172.2) of Ala; N-CH (δ : 2.97 (3H, s) of N-Me-Val to C=O (δ
68.3) of N-Me-Cys)), indicated that the connections were Dehydroxy-Ser-Ala′-N-Me-Cys′-N-Me-Val′-
H
: 4.85 (1H, m) of Ala′ to the C=O (δ
C
: 163.2) of Dehydroxy-Ser; N-CH
(δ : 3.02 (3H, s) of N-Me-Val′ to the
: 169.9) of
(δ : 3.15
3
(δ
H
:
3
C
: 172.2) of Ala′; N-CH
3
H
C
H
C
H
C
3
H
(
C
3
H
C
:
1
Ser-Ala-N-Me-Cys-N-Me-Val. Above all evidence, compound 1 was structurally similar to
echinomycin. The only difference between them was the presence of a double bond (δ : 6.90 (1H, brs),
: (133.0, 104.3)) in compound 1. In the HMBC spectrum, the methylene protons (δ
.90 (1H, brs), 6.11 (1H, brs)) to C=O (δ : 163.2), confirmed that the double bond originated from the
H
6
6
.11 (1H, brs); δ
C
H
:
C
Ser. On the basis of the above information, all protons and carbon resonances were assigned and the
planar structure of compound 1 was established. Because the planar differences in the structures of
compound 1 and echinomycin cause the changes on spatial configurations, the NMR spectral data,
1
especially H NMR spectral data of compound 1 were different with that of echinomycin. The
appearance of double bond of Dehydroxy-Ser may make the quinoxaline, amide, alkene, and carbonyl
groups form a large conjugate plane (Supplementary Materials Figure S9). The CH
3
of the Ala′
1
positioned in the shielding area, so its H NMR spectral data upfielded to δ
H
: 0.19.

Mar. Drugs 2015, 13
6950
1
13
Table 1. H (500 MHz) and C (125 MHz) NMR Date for Compound 1 (CDCl ).
3
Position
Quinoxaline
2
δ
H
, mult (J in Hz)
δ
C
Position
δ
H
, mult (J in Hz)
δ
C
Position
δ
H
, mult (J in Hz)
δ
C
β
5.02, dd (11.5, 3.0)
4.67, d (11.5)
65.8
α
β
5.28, m
45.6
18.3
143.9
143.9
143.5
143.7
129.4
129.4
132.4
131.9
131.6
131.0
130.0
130.0
140.3
140.3
143.4
142.4
163.6
161.9
1.35, d (6.0)
2
′
C=O
169.9
C=O
Ala′
NH
α
172.2
3
9.68, s
9.63, s
Dehydroxy-Ser
a
3
′
NH
10.67, s
7.83
5
8.20, d (8.0)
8.20, d (8.0)
α
133.0
104.3
163.2
4.85, m
44.9
16.4
5
′
β
6.90, brs; 6.11, brs
β
0.19, d (6.0)
a
6
7.84–7.89
C=O
C=O
172.2
a
6
′
7.84–7.89
N-Me-Cys
N-Me-Val
a
7
7.95–7.97
N-Me
3.15, s
5.70, s
33.2
60.4
52.1
13.4
168.3
N-Me
2.97, s
3.42, d (10.5)
2.27, m
29.0
65.8
27.4
19.5
18.5
169.8
a
7
′
7.95–7.97
α
α
8
8.27, d (8.0)
β
S-Me
C=O
N-Me-Cys′
N-Me
α
3.74, d (2.0)
2.07,s
β
a
a
8
′
8.20, d (8.0)
γ
1.04
9
γ
0.97, d (6.0)
9
′
COOH
1
0
3.37, s
33.8
61.1
29.2
N-Me-Val′
1
0′
6.03, d (4.0)
N-Me
3.02, s
28.8
64.6
28.6
19.7
18.8
169.9
C=O
C=O′
Ser
β
3.47, dd (16.0, 5.0)
2.54, d (16.0)
α
4.47, d (11.0)
2.49, m
β
γ
C=O
Ala
171.5
1.06, d (7.0)
1.03, d (7.0)
NH
α
9.01, d (9.5)
5.37, d (9.0)
γ
52.6
NH
9.20, d (9.5)
C=O
a
Overlapping signals.

Mar. Drugs 2015, 13
6951
Figure 2. Key HMBC and ROESY correlations of compound 1.
Marfey’s method was employed to assign the absolute configurations of the amino acid residues
resulting from acid hydrolysis of 1 [15,16]. The 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA)
derivatives of the acid hydrolysate of 1 and the authentic D- and L-amino acids were subjected to
HPLC analysis. The absolute configurations of all amino acid residues in 1 except for N-Me-Cys were
established by comparing their HPLC retention times with those of the corresponding authentic D- and
L- amino acid standards (Table 2, Supplementary Materials Figure S17). The ROESY correlation
between H-α of N-Me-Cys (δ
H
: 5.70) and H-α of N-Me-Cys′ (δ : 6.03) showed they necessarily
H
positioned cis to each other. In other words, these two N-Me-Cys were either D or L amino acids. The
absolute configuration of the N-Me-L-Cys was confirmed by the ROESY correlation between H-α of
N-Me-Cys (δ
observed implying H-α of N-Me-Cys (δ
: 0.97). The results indicated that the amino acid residues were L-Ala/Ala′, D-Ser, N-Me-L-Val/N-
H H
: 5.70) and H-γ of N-Me-L-Val (δ : 0.97) (Figure 2), because this correlation was
H
: 5.70) located at β-orientaion as same as H-γ of N-Me-L-Val
(
δ
H
Me-L-Val′ and N-Me-L-Cys/N-Me-L-Cys′ configurations.
Table 2. The retention times of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA)
derivatives of the hydrolysates of 1–2 and amino acids standards.
Retention Time (T ) Min
R
Amino Acid Standards
15.787
Compound 1
Compound 2
L-Ser
D-Ser
-
-
16.422
16.649
-
L-Ala
22.125
21.952
-
D-Ala
23.789
-
-
N-Me-L-Val
N-Me-D-Val
L-Pro
17.499
17.528
-
19.732
-
-
14.544
-
-
14.677
-
D-Pro
17.528
FDAA
19.772
19.804
19.782
Thus, as shown in 1 (Figure 1), the absolute stereochemistry of this novel echinomycin analogue
was assigned and it was given the trivial name quinomycin G.
Cyclo(L-Pro-4-OH-L-Leu) (2) was isolated as a white powder, its molecular formula C11
H
18
N
2
O
3
+
was established upon the analysis of the HRESIMS peak at m/z 249.1209 [M + Na] , indicating four

Mar. Drugs 2015, 13
6952
1
degrees of unsaturation. The H NMR spectrum of 2 (Table 3) revealed one NH resonance (δ
1H, s)); two methine signals (δ
resonances (δ : 3.56 (2H, m); 2.01 (1H, m), 1.89 (1H, m); 2.36 (1H, dd, J = 8.5, 2.5 Hz), 2.10 (1H, m);
.32 (1H, dd, J = 14.5, 2.0 Hz), 1.79 (1H, dd, J = 14.5, 11.0 Hz)); two methyl signals in the upfield
H
: 7.66
(
H
: 4.23 (1H, d, J = 10.5 Hz), 4.08 (1H, t, J = 8.0 Hz)); four methylene
H
2
1
3
region (δ
H
: 1.31 (1H, s), 1.35 (1H, s)). The C NMR spectra of 2 (Table 3) showed a total of 11 carbon
: 169.7, 166.1), three methines (δ
: 45.6, 41.1, 28.3, 22.6), two methyl signals (δ : 32.3,
7.4). Further analysis of the NMR data confirmed the existence of moieties of Pro and 4-hydroxyl-Leu.
: 3.56 (2H, m)), and H-9 (δ : 4.23 (1H,
: 2.36 (1H, dd, J = 8.5, 2.5 Hz), 2.10 (1H, m)) and H-8
: 58.8) confirmed the planar structure for compound 2 was
cyclo(Pro-4-hydroxyl-Leu). The relative stereochemistry of 2 was deduced from the NOE spectrum
Figure 3). When irradiating H-6 at δ : 4.08 and H-9 at δ : 4.23, the integration values of H-9 and H-6
resonances, and they were classified as two amide carbonyls (δ
C
C
:
7
2
0.9, 58.8, 53.2), four methylene signals (δ
C
C
In the HMBC experiment (Figure 3), the correlations of H-3 (δ
d, J = 10.5 Hz)) with C-1 (δ : 166.1); H-5 (δ
: 7.66 (1H, s)) with C-6 (δ
H
H
C
H
(
δ
H
C
(
H
H
were enhanced respectively, which showed that H-6 and H-9 positioned cis to each other. That is to
say relative configurations of Pro and 4-hydroxyl-Leu were either L or D configurations. On the basis
of Marfey’s method, the presence of L-Pro in 2 compared with the appropriate amino acid standards
(
Table 2, Supplementary Materials Figure S17), determined the configuration of compound 2
as cyclo(L-Pro-4-hydroxyl-L-Leu).
1
13
Table 3. H (500 MHz) and C (125 MHz) NMR Date for compound 2 (CDCl
3
).
No.
1
δ
H
, mult (J in Hz)
δ
C
No.
7
δ
H
, mult (J in Hz)
δ
C
166.1
169.7
2
8
7.66, s
3
3.56, m
1.89, m
2.01, m
45.6
9
4.23, d (10.5)
53.2
41.1
4α
4β
5α
5β
10α
10β
11
12
13
2.32, dd (14.5, 2.0)
1.79, dd (14.5, 11.0)
2
2
2.6
8.3
2.36, dd (8.5, 2.5)
2.10, m
70.9
27.4
32.3
1.31, s
1.35, s
6
4.08, t (8.0)
58.8
Figure 3. The Key HMBC and NOE correlations of compound 2.
In our ongoing investigation of active metabolites, tirandamycins A (3) [17] and B (4) [18] were
obtained and their structures were elucidated by the MS, H, 13C NMR, HSQC, HMBC spectral
analysis (Supplementary Materials Figure S27) and comparison of the respective spectral data with
1
1
13
those found in the literature. Surprisingly, their H and C NMR spectral data showed significant
differences at positions 1, 2, 3 and 5 (Table 4), though they were tested in the same solvent (DMSO-d ).
6

Mar. Drugs 2015, 13
6953
Subsequently, this inspired us to study the structural distinction between them. The literature survey
indicated that the substituent groups on bicyclic ketal moiety have little influence on the NMR spectral
data of the long conjugated system [17–22]. Therefore, we proposed that the distinct differences of
NMR spectral data were caused by the positions of the enolic hydroxy and carbonyl group. Compared with
C-1 at δ
C
: 173.5 in tirandamycin A (3), the data of C-1 in tirandamycin B (4) moved to the downfield
at δ : 181.0, implying that 4 should be in 1-keto-4′-enol form. The keto-enol tautomer existed widely
C
in the structure of natural products, and the rules of NMR data of these two tautomers have been
studied [23], which also supported that tirandamycin B (4) was the 1-keto-4′-enol form. It is the
first time to reveal the 1-keto-4′-enol form of tirandamycin B explicitly. Because the structures of
1
-keto-4′-enol form of tirandamycins were unclear previously, the assignments of the NMR data of
these compounds were not correct [21,22]. Herein, we summarized the trend in NMR data of
keto-enol tautomers exist in tirandamycins in order to raise concern on the structural and NMR
data differences between these two forms. In the 13C NMR spectrum, when the structure was in
1
-enol-4′-keto form just like tirandamycin A, the carbon signals occurred at approximately δ
C
: 173.5
(
C-1), 116.2 (C-2), 147.9 (C-3) and 143.7 (C-5), however the carbons of the 1-keto-4′-enol form as
tirandamycin B resonated at approximately δ
C
: 181.0 (C-1), 124.8 (C-2), 143.2 (C-3) and 137.9 (C-5).
1
More importantly, three olefinic protons had obvious differences in these two tautomers in the H NMR
spectrum, δ
H
: 7.05 (H-2) 7.47 (H-3) and 6.19 (H-5) in 1-enol-4′-keto form changed to δ
H
: 7.55 (H-2)
7
.14 (H-3) and 5.81 (H-5) in 1-keto-4′-enol form. The chemical shift of H-2 at δ
H
: 7.55 increased
abnormally, which may be due to the shielding effect of the double bond (1C=O). According to the
1
above results and literature survey [17–22,24], we also summed up a brief rule that if the H NMR data
H
of H-5 is more than δ : 6.00, the structure of tirandamycin is in 1-enol-4′-keto form, otherwise, it is in
the other form.
1
13
Table 4. The H (500 MHz) and C (125 MHz) NMR Date for Tirandamycins A and
B (DMSO-d ).
6
Tirandamycin A
, mult (J in Hz)
Tirandamycin B
, mult (J in Hz)
No.
δ
H
δ
C
δ
H
δ
C
1
2
3
4
5
6
7
8
9
173.5
116.2
147.9
134.1
143.7
33.9
181.0
124.8
143.2
134.2
137.9
33.5
7.05, d (15.5)
7.47, d (15.5)
7.55, d (15.5)
7.14, d (15.5)
6.19, d (9.5)
2.88, m
5.81, d (10.0)
2.83, m
3.77, dd (9.0, 2.0)
1.81, m
75.8
3.73, m
76.1
33.9
1.77, m
33.7
4.03, d (6.5)
77.8
4.04, d (6.5)
77.7
1
1
1
1
0
1
2
3
202.8
59.9
202.5
56.0
3.47, s
3.56, s
56.7
59.8
96.2
95.3

Mar. Drugs 2015, 13
6954
Table 4. Cont.
Tirandamycin A
, mult (J in Hz)
Tirandamycin B
, mult (J in Hz)
No.
δ
H
δ
C
δ
H
δ
C
1
1
1
1
4
5
6
7
1.44, s
1.84, s
22.4
12.0
16.6
11.0
1.43, s
1.79, s
23.3
12.5
16.9
11.1
1.07, d (7.0)
0.62, d (7.0)
1.05, d (6.5)
0.62, d (6.5)
3
3
.84, dd (12.5, 3.5)
.74, dd (12.5, 3.5)
7.58, s
1
8
1.40, s
8.74, s
15.1
56.5
1
′
′
′
′
2
176.0
100.7
193.9
177.9
101.2
192.9
3
4
3
3
.77, dd (9.0, 2.0)
.75, dd (9.0, 2.0)
5
′
51.4
3.47, s
50.2
1
8-OH
5.02 brs
1
The variable temperature experiments for H NMR of tirandamycins A (3) and B (4) were
employed for studying the tautomerizm of keto-enol tirandamycins and the test temperatures were set
at 40, 60, 80 °C. With the increase of test temperature, the structure of tirandamycin A (3) was still in
the 1-enol-4′-keto form (Supplementary Materials Figure S21), but the structure of tirandamycin B (4)
gradually transformed to 1-enol-4′-keto form (Supplementary Materials Figure S26). The results
suggested that in the DMSO-d solution, tirandamycin A (3) was stable in 1-enol-4′-keto form, while
6
tirandamycin B (4) was more stable in 1-keto-4′-enol form than the other form. The reason may be the
structure itself or external factors, which need to be further investigated.
The known antibiotic staurosporine (5) was characterized by comparison of the respective spectral
1
13
data (MS, H, C NMR) with those found in the literature [25].
2
.2. Biological Assays
The novel echinomycin analogue compound 1 was assayed for antibacterial activities against
Staphylococcuse pidermidis (ATCC 12228 (MSSE), 12-6(MSSE), 12-8(MRSE)), Staphylococcus
aureus (ATCC 29213(MSSA), ATCC 33591(MRSA), 15(MSSA), 12-28(MSSA), 12-33(MRSA)),
Enterococcus faecium (ATCC 700221(VRE), 12-1(VRE), 12-3(VSE)), and Enterococcus faecalis
(ATCC 292121(VSE), ATCC 51299 (VRE), 12-5(VSE), 09-9 (VRE)) and compared with the controls,
echinomycin and levofloxacin. Compound 1 exhibited moderate antibacterial activities with MIC
values of 16–64 μg/mL against not only the antibiotic-sensitive but also the antibiotic-resistant
bacteria. Its activities were lower than those of echinomycin (Table 5).
Meanwhile, compound 1 was also evaluated for anti-tumor activity in vitro against 12 human tumor
cell lines (HCT-116, HepG2, BGC-823, NCI-H1650, A2780, SW1990, Mia-PaCa-2, U87 MG, SK-N-SH,
Jurkat, ACHN, 786-O) by MTT method and echinomycin was used as the control. Compound 1
displayed strong anti-tumor activity against the tested cell lines ACHN, 786-O, U87 MG and Jurkat
with the IC50 values of 0.552, 0.721, 0.627, and 0.414 μM, respectively. Unfortunately, its activities
were also lower than those of echinomycin (Table 6).

Mar. Drugs 2015, 13
6955
Table 5. Minimum inhibitory concentrations (MIC) for compound 1 and echinomycin.
MIC (μg/mL)
Microorganism
Strain No.
Staphylococcus epidermidis
Staphylococcus aureus
Enterococcu faecium
Enterococcus faecalis
ATCC
ATCC
29213
MSSA c
32
ATCC
ATCC
ATCC
ATCC
51299
VRE
16
12-6
12-8
15
12-28
12-33
12-1
12-3
12-5
09-9
1
2228
33591
700221
29212
VSE
16
Phenotype
Compound 1
MSSE a
MSSE
32
MRSE b
MRSA d MSSA MSSA MRSA VRE e VRE VSE f
VSE VRE
32
32
0.5
4
32
0.5
32
0.5
32
32
0.5
64
32
0.5
128
32
0.5
128
64
0.5
64
16
0.25
1
16
Echinomycin
0.5
0.25
0.5
0.5
0.25
0.25
0.25
0.5
0.5
0.25
>128
Levofloxacin (conrol)
0.25
0.125
0.25
0.125
1
Notes: a Methicillin-susceptible Staphylococcus epidermidis; b Methicillin-resistant Staphylococcus epidermidis; c Methicillin-susceptible Staphylococcus aureus;
d
e
f
Methicillin-resistant Staphylococcus aureus; Vancomycin-susceptible Enterococcus; Vancomycin-resistant Enterococcus.
Table 6. Anti-tumor activities (IC50, μM) of compound 1 and echinomycin.
IC50 (μM)
Compounds
ACHN SW1990 Mia-PaCa-2
786-O
U87 MG SK-N-SH Jurkat HCT-116 NCI-H1650 HepG2 BGC-823 A2780
Compound 1
0.552
2.560
4.750
0.721
0.627
5.174
0.027
0.414
8.61
3.90
>10
>10
>10
Echinomycin <0.0032
0.0026
<0.0032
<0.0032 <0.0032
<0.0032
<0.01
<0.01
<0.01
<0.01
<0.01

Mar. Drugs 2015, 13
6956
3. Experimental Section
3.1. General
Optical rotations were measured on a JASCO P-2000 digital polarimeter (JASCO Corporation
Tokyo, Japan). UV measurements were recorded using an Agilent Cary 300 spectrometer (Agilent
1
13
Technologies, Santa Clara, United States). H and C NMR, and 2D NMR spectra were obtained at
00 and 125 MHz, using a Bruker AVANCE 500-III spectrometer (Bruker Biospin Group, Karlsruhe,
Germany) in chloroform or DMSO-d with TMS as an internal reference. HR-ESI-MS data were
5
6
measured using an Agilent 1100 LC/MSD Trap SL LC/MS/MS spectrometer (Agilent Technologies,
Santa Clara, CA, USA). Semipreparative HPLC was performed by an HPLC system equipped with a
Shimadzu LC-6AD pump and a Shimadzu SPD-20A prominence UV-VIS detector (Shimadzu
Corporation, Kyoto, Japan) using an Agilent C18 column (5 μM, 250 × 9.6 mm). Column chromatography
was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China),
RP-18 (40–60 μM, GE healthcare, Fairfield, CT, USA), and Sephadex LH-20 (18–110 μM, GE healthcare,
Fairfield, CT, USA)
3
.2. Bacterial Material and Fermentation
The producing strain LS298 was isolated from a sponge Gelliodes carnosa collected from Lingshui
Bay, Hainan Province of China near Xincun Harbor (18°24′5.49″ N, 109°59′37.76″ E), in August 2007 [9].
It was identified as Streptomyces sp. on the basis of the morphology and 16S rRNA gene sequence
analysis by comparison with other sequences in the GenBank database. The DNA sequence was
deposited in GenBank (Accession No. FJ937945). The strain LS298 was first cultivated on Gause I
agar plates (Gause I: starch 20 g; KNO
3
1 g; NaCl 0.5 g; K
2
HPO
3
0.5 g; MgSO 0.01 g; Natural
4
seawater 1 L; pH 7.0–7.2) at 28 °C for three days. Then, the mycelia were inoculated into 500 mL
Erlenmeyer flasks, each containing 100 mL of liquid A1 medium (A1: starch 10 g; Yeast extract 4 g;
Peptone 2 g; Natural seawater 1 L; pH 7.0–8.0). The flasks were incubated at 28 °C on a rotary shaker
(
200 rpm) for three days. Seed culture (10 mL) was transferred into three hundred 500 mL Erlenmeyer
flasks (each Erlenmeyer flask contained 100 mL A1 medium) and incubated at 28 °C on a rotary
shaker (200 rpm) for nine days.
3.3. Extraction and Isolation
The culture broth (30 L) was repeatedly extracted with ethyl acetate (v/v 1:3, three time) by
ultrasound, and the organic solvent was evaporated to dryness under a vacuum to afford the crude
extract (3.5 g). The crude extract was first subjected to reversed-phase (RP) column chromatography
(
4 × 60 cm, 200 g) using a decreasing polar aqueous CH
each 2 L) gradient elution and afforded four primary fractions (A1–A4). Fraction A4 (100% CH
20 mg) was separated again by Sephadex LH-20 chromatography (2 × 100 cm, 80 g) using 1:10
CHCl /CH OH (each 20 mL) as eluent. Then, the fractions were combined to four fractions (B1–B4)
on the basis of their HPLC behavior. Purification of the B2 subfractions (100 mg) by semipreparative
HPLC afforded compound 1 (10.0 mg, 55% CH CN in H O, flow rate 4 mL/min, t = 37.8 min).
3
OH/H
2
O (20%, 50%, 80%, and 100%, v/v,
3
OH,
9
3
3
3
2
R

Mar. Drugs 2015, 13
6957
Fraction A1 (20% CH
3
OH/H
2
O, 715 mg) was separated via Sephadex LH-20 (2 × 100 cm, 80 g) using
CH
were combined, then it was purified by semipreparative HPLC to provide compound 2 (8.2 mg, 36%
CH CN in H O, flow rate 4 mL/min, t = 27.3 min). Fraction A3 (80% CH OH/H O, 530 mg) was
further separated on RP chromatography (3 × 40 cm, 100 g) eluted with CH OH/H O (60%, 70%,
0%, 100%, v/v, each 1 L) and fraction A3.3 (80% CH OH, 50 mg) was purified by Sephadex LH-20
chromatography (2 × 100 cm, 80 g) eluted with CH OH to get the yellow band compound 3 (7.5 mg).
Fraction A2 (50% CH OH/H O, 450 mg) was further separated by semipreparative HPLC to provide
compounds 4 (8.0 mg, 42% CH CN in H O, flow rate 4 mL/min, t = 31.5 min)) and 5 (7.8 mg, 47%
CH CN in H O, flow rate 4 mL/min, t = 42.6 min).
Compound 1: amorphous yellow powder; [α] −3.8 (c 0.20, CHCl
3
OH (each 20 mL) as eluent. The fractions were examined by HPLC and the Fraction A1.5 to A1.8
3
2
R
3
2
3
2
8
3
3
3
2
3
2
R
3
2
R
2
D
5
3
); UV (CHCl
3
) λmax (log ε) 245.2
1
13
+
(
2.6), 325.8 (1.9); H and C NMR data, see Table 1; HRESIMS m/z 1101.4288 [M + H] (calcd for
C
51
H
65
N
12
O
12
S
2
, 1101.4286).
2
D
5
1
Compound 2: white powder; [α] −15.0 (c 0.20, CH
13
3
OH); UV (CH
3
OH) λmax (log ε) 240.2 (3.6); H
Na, 249.1210).
+
and C NMR data, see Table 3; HRESIMS m/z 249.1209 [M + Na] (calcd for C11
H
18
N
2
O
3
3
.4. Hydrolysis of Compounds 1–2 and HPLC Analysis by Marfey’s Method
Compounds 1 (1.0 mg) and 2 (1.4 mg) were dissolved in 6 N HCl (1 mL), and heated at 110 °C for
1
8 h. After cooling to room temperature, the hydrolysates were dried under reduced pressure and
resuspended into 100 μL of H
2
O.Then they were treated with 1 M NaHCO (25 μL), and reacted with
3
1
00 μL of 1% (w/v) FDAA in acetone at 40 °C for 1.5 h. After cooling to room temperature, the
mixture was added with 1 M HCl (25 μL) to neutralize and terminate the reaction. MeOH was then
added to the quenched reaction to afford a total volume of 500 μL; 10 μL of each hydrolysate
derivatization reaction was used for HPLC analysis using an Agilent C18 column (150 × 4.6 mm,
5
μM) with a solvent gradient from 15% to 45% solvent B (solvent A: CH
solvent B: CH CN) over the course of 30 min and UV detection at 340 nm at a flow rate of 1 mL/min.
Similarly, 10 μL of the standard amino acids in H O (4 μM) were added to 1 M NaHCO (20 μL) and
3
COOH/H
2
O, 0.05/99.95,
3
2
3
each mixture was treated with 1% (w/v) FDAA (50 μL) for 1.5 h at 40 °C. Derivatization reactions
were terminated with 1 M HCl (20 μL) and diluted to a total volume of 500 μL with MeOH. Of these
standard amino acid derivatization reactions, 10 μL was subjected to HPLC analysis and used as
structural standards in the elucidation of structures 1 and 2.
3
.5. Biological Assays
Antibacterial and anti-tumor assays were performed with compounds of purity >90% by HPLC.
.5.1. Antibacterial Activity
3
The testing bacteria used in this study were as follows: Staphylococcus epidermidis (ATCC 12228
(
MSSE), 12-6 (MSSE), 12-8 (MRSE)), Staphylococcus aureus (ATCC 29213 (MSSA), ATCC 33591
MRSA), 15 (MSSA), 12-28 (MSSA), 12-33 (MRSA)), Enterococcus faecium (ATCC 700221 (VRE),
(
1
2-1 (VRE), 12-3 (VSE)), Enterococcus faecalis (ATCC 292121 (VSE), ATCC 51299 (VRE), 12-5

Mar. Drugs 2015, 13
6958
(
VSE), 09-9 (VRE)), which included strains from the ATCC collection and clinical isolates. MIC
values against the 15 bacterial strains for compound 1 were measured by using the agar dilution
method described by the Clinical Laboratory Standards Institute [26]. Briefly, the test medium was
Mueller-Hinton broth, and the inoculum was 10,000 colony forming units (CFU)/spot. The compound
1
was incorporated into the agar medium, with each plate containing a different concentration of
the compound. Culture plates were incubated at 35 °C for 18 h, and MICs were then recorded.
The positive controls were levofloxacin and echinomycin. The final concentrations of compounds
ranged from 0.03 to 128 μg/mL. The MIC was defined as the lowest concentration that prevented
visible growth of the bacteria [27].
3
.5.2. Anti-Tumor Assay
The anti-tumor activities of the new compound 1 and echinomycin against 12 tumor cell lines
including: human colonic carcinoma (HCT-116), human hepatoma (HepG2), human gastric cancer
BGC-823), human non-small cell lung cancer (NCI-H1650), human ovarian cancer (A2780), human
(
pancreatic cancer (SW1990, Mia-PaCa-2), human multiform glioblastoma (U87 MG), Human
neuroblastoma (SK-N-SH), human T-cell leukemia (Jurkat) and human renal clear cell carcinoma
(
ACHN, 786-O) were determined by MTT method [28–31].
The human colonic carcinoma (HCT-116), human hepatoma (HepG2), human gastric cancer
BGC-823), human non-small cell lung cancer (NCI-H1650), human ovarian cancer (A2780), human
(
pancreatic cancer (SW1990, Mia-PaCa-2), human multiform glioblastoma (U87 MG), human
neuroblastoma (SK-N-SH), human renal clear cell carcinoma (ACHN, 786-O) were maintained in
DMEM medium; human T-cell leukemia (Jurkat) was maintained in RPMI 1640 medium. Both media
were supplemented with 10% heat inactivated fetal bovine serum, 100 units/mL of penicillin and
1
00 μg/mL of streptomycin, in a humidified 5% CO /air atmosphere at 37 °C. MTT assay: briefly,
2
logarithmic cells were digested with 0.25% pancreatic enzyme-EDTA and plated in the 96-well plates
at concentration of 800–2000/100 μL per well. Compounds at final concentrations of 0.5 to 50 μg/mL
were added with triplicates of each concentration after 24 h. The cells were incubated further at 37 °C
for 96 h, the medium was aspirated, and 100 μL MTT of 0.5 mg/mL in medium was added. After 4h
incubation, the medium was aspirated and 200 μL DMSO was added to solubilize the formazan
crystals. Absorbance of the converted dye was measured at a wavelength of 570 nm with background
subtraction at 650 nm. The dose-response curves were fitted with Sigma plot and IC50s were determined.
4
. Conclusions
In summary, a novel echinomycin analogue quinomycin G (1), a new cyclic dipeptide cyclo-(L-Pro-
-OH-L-Leu) (2), along with three known antibiotics tirandamycin A (3), tirandamycin B (4) and
4
staurosporine (5), were isolated and characterized from the marine Streptomyces sp. LS298. To our
knowledge, this was the first time to obtain three types of antibiotics from one strain from the same batch,
though these types of antibiotics have been isolated from different strains of genus Streptomyces [18,32].
What is more, the 1-keto-4′-enol form of tirandamycin B was reported firstly, and the trend in NMR
data of keto-enol tautomers in tirandamycins was discussed.

Mar. Drugs 2015, 13
6959
Compound 1 exhibited moderate antibacterial and remarkable anti-tumor activities; however, its
activities were lower than those of echinomycin, which indicated that the bicyclic peptide of these
compounds was required for their activities. Echinomycin has been studied for many years and the
mechanism of its antibacterial and antitumor activities is considered to be DNA bis-intercalation.
Due to the similar structure, we proposed that compound 1 has a similar mechanism against the
bacteria and the tumor cells. Efforts are underway to discover novel and potential echinomycin analogues
in future work through combinations of genome mining and heterologous expression approaches.
Acknowledgments
This work was financially supported by grants from the Mega-Project for Innovative Drugs (grant
number 2012ZX09301002-001-005) and the National Natural Science Foundation of China (grant
number 81402846). We are grateful to the Department of Instrumental Analysis of our Institute for
measurement of the IR, NMR, and MS data. We acknowledge the research Team of Xue-fu You,
Department of Pharmacology, Institute of Medicinal Biotechnology, and the Chinese Academy of
Medical Sciences for the antibacterial activity bioassays.
Author Contributions
X.Z., T.G. and P.Z. conceived and designed the experiments; X.Z., T.G. and F.L. performed the
experiments; X.Z., T.G., F.L. and P.-C.Z. analyzed the data; W.-Q.Z. and Y.L. contributed the
anti-tumor biassays; X.Z., T.G. and P.Z. wrote the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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