Next Article in Journal
Salternamide A Suppresses Hypoxia-Induced Accumulation of HIF-1α and Induces Apoptosis in Human Colorectal Cancer Cells
Next Article in Special Issue
Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes
Previous Article in Journal
Cyanobactins from Cyanobacteria: Current Genetic and Chemical State of Knowledge
Previous Article in Special Issue
Structure Elucidation and in Vitro Toxicity of New Azaspiracids Isolated from the Marine Dinoflagellate Azadinium poporum
Open AccessArticle

A New Analogue of Echinomycin and a New Cyclic Dipeptide from a Marine-Derived Streptomyces sp. LS298

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
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editor: Orazio Taglialatela-Scafati
Mar. Drugs 2015, 13(11), 6947-6961;
Received: 2 July 2015 / Revised: 5 November 2015 / Accepted: 6 November 2015 / Published: 18 November 2015
(This article belongs to the Special Issue Marine Secondary Metabolites)


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 12 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 marine-derived Streptomyces; secondary metabolites; antibacterial activity; anti-tumor activity

1. Introduction

With the emergence of newer resistant forms of infectious diseases and multi-drug resistant (MDR) 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 [2,3,4]. The immense diversity of marine actinomycetes, along with their underutilization, has attracted great attention from researchers to discover novel antibiotics [5,6,7,8].
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 [11,12,13,14]. 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.
Figure 1. Structures of the isolated compounds 15 and reference compound echinomycin.
Figure 1. Structures of the isolated compounds 15 and reference compound echinomycin.
Marinedrugs 13 06947 g001

2. Results and Discussion

2.1. Structure Elucidation of Compounds 15

Quinomycin G (1) was obtained as an amorphous yellow powder, a molecular formula of C51H64N12O12S2 was determined by HRESIMS (m/z 1101.4288 [M + H]+, calcd for C51H65N12O12S2, 1101.4286), 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]. The 1H NMR spectrum of 1 (Table 1) displayed four NH resonances (δH: 10.67 (1H, s), 9.20 (1H, d, J = 9.5 Hz), 9.01 (1H, d, J = 9.5 Hz), 7.83 (1H, overlap)); 12 aromatic protons signals (δH: 9.68 (1H, s), 9.63 (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 (δH: 5.02 (1H, dd, J = 11.5, 3.0 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)); ten methine signals (δH: 6.03 (1H, d, J = 4.0 Hz), 5.70 (1H, s), 5.37 (1H, d, J = 9.0 Hz), 5.28 (1H, m), 4.85 (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), 2.27 (1H, m)); 11 methyl signals in the upfield region, including four N-Me groups (δH: 3.37 (3H, s), 3.15 (3H, s), 3.02 (3H, s), 2.97 (3H, s)), one S-Me group (δH: 2.07 (3H, s)). 51 carbons were observed in the 13C NMR spectrum of compound 1 (Table 1), including ten ester/amide carbonyls (δC: 172.2 (2C), 171.5, 169.9 (2C), 169.8, 168.3, 163.6, 163.2, 161.9) and 18 sp2 carbon signals (δC: 143.9 (2C), 143.7, 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), 104.3). Comprehensive analysis of the 1H-1H 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 (δH: 4.85 (1H, m) of Ala′ to the C=O (δC: 163.2) of Dehydroxy-Ser; N-CH3H: 3.37 (3H, s) of N-Me-Cys′ to the C=O (δC: 172.2) of Ala′; N-CH3H: 3.02 (3H, s) of N-Me-Val′ to the C=O (δC: 171.5) of N-Me-Cys′; H-β (δH: 4.67 (1H, d, J = 11.5 Hz) of Ser to the C=O (δC: 169.9) of N-Me-Val′; NH (δH: 9.20 (1H, d, J = 9.5 Hz) of Ala to the C=O (δC: 169.9) of Ser; N-CH3H: 3.15 (3H, s) of N-Me-Cys to the C=O (δC: 172.2) of Ala; N-CH3H: 2.97 (3H, s) of N-Me-Val to C=O (δC: 168.3) of N-Me-Cys)), indicated that the connections were Dehydroxy-Ser-Ala′-N-Me-Cys′-N-Me-Val′-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 (δH: 6.90 (1H, brs), 6.11 (1H, brs); δC: (133.0, 104.3)) in compound 1. In the HMBC spectrum, the methylene protons (δH: 6.90 (1H, brs), 6.11 (1H, brs)) to C=O (δC: 163.2), confirmed that the double bond originated from the 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, especially 1H 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 CH3 of the Ala′ positioned in the shielding area, so its 1H NMR spectral data upfielded to δH: 0.19.
Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Date for Compound 1 (CDCl3).
Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Date for Compound 1 (CDCl3).
PositionδH, mult (J in Hz)δCPositionδH, mult (J in Hz)δCPositionδH, mult (J in Hz)δC
Quinoxaline β5.02, dd (11.5, 3.0)65.8α5.28, m45.6
2 143.9 4.67, d (11.5) β1.35, d (6.0)18.3
2′ 143.9C=O 169.9C=O 172.2
39.68, s143.5Dehydroxy-Ser Ala′
3′9.63, s143.7NH10.67, s NH7.83 a
58.20, d (8.0)129.4α 133.0α4.85, m44.9
5′8.20, d (8.0)129.4β6.90, brs; 6.11, brs104.3β0.19, d (6.0)16.4
67.84–7.89 a132.4C=O 163.2C=O 172.2
6′7.84–7.89 a131.9N-Me-Cys N-Me-Val
77.95–7.97 a131.6N-Me3.15, s33.2N-Me2.97, s29.0
7′7.95–7.97 a131.0α5.70, s60.4α3.42, d (10.5)65.8
88.27, d (8.0)130.0β3.74, d (2.0)52.1β2.27, m27.4
8′8.20, d (8.0) a130.0S-Me2.07,s13.4γ1.04 a19.5
9 140.3C=O 168.3γ0.97, d (6.0)18.5
9′ 140.3N-Me-Cys′ COOH 169.8
10 143.4N-Me3.37, s33.8N-Me-Val′
10′ 142.4α6.03, d (4.0)61.1N-Me3.02, s28.8
C=O 163.6β3.47, dd (16.0, 5.0)29.2α4.47, d (11.0)64.6
C=O′ 161.9 2.54, d (16.0) β2.49, m28.6
Ser C=O 171.5γ1.06, d (7.0)19.7
NH9.01, d (9.5) Ala γ1.03, d (7.0)18.8
α5.37, d (9.0)52.6NH9.20, d (9.5) C=O 169.9
a Overlapping signals.
Figure 2. Key HMBC and ROESY correlations of compound 1.
Figure 2. Key HMBC and ROESY correlations of compound 1.
Marinedrugs 13 06947 g002
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 (δH: 5.70) and H-α of N-Me-Cys′ (δH: 6.03) showed they necessarily 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 (δH: 5.70) and H-γ of N-Me-l-Val (δH: 0.97) (Figure 2), because this correlation was observed implying H-α of N-Me-Cys (δH: 5.70) located at β-orientaion as same as H-γ of N-Me-l-Val (δH: 0.97). The results indicated that the amino acid residues were l-Ala/Ala′, d-Ser, N-Me-l-Val/N-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 12 and amino acids standards.
Table 2. The retention times of 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (FDAA) derivatives of the hydrolysates of 12 and amino acids standards.
Retention Time (TR) Min
Amino Acid StandardsCompound 1Compound 2
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 C11H18N2O3 was established upon the analysis of the HRESIMS peak at m/z 249.1209 [M + Na]+, indicating four degrees of unsaturation. The 1H NMR spectrum of 2 (Table 3) revealed one NH resonance (δH: 7.66 (1H, s)); two methine signals (δH: 4.23 (1H, d, J = 10.5 Hz), 4.08 (1H, t, J = 8.0 Hz)); four methylene resonances (δH: 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); 2.32 (1H, dd, J = 14.5, 2.0 Hz), 1.79 (1H, dd, J = 14.5, 11.0 Hz)); two methyl signals in the upfield region (δH: 1.31 (1H, s), 1.35 (1H, s)). The 13C NMR spectra of 2 (Table 3) showed a total of 11 carbon resonances, and they were classified as two amide carbonyls (δC: 169.7, 166.1), three methines (δC: 70.9, 58.8, 53.2), four methylene signals (δC: 45.6, 41.1, 28.3, 22.6), two methyl signals (δC: 32.3, 27.4). Further analysis of the NMR data confirmed the existence of moieties of Pro and 4-hydroxyl-Leu. In the HMBC experiment (Figure 3), the correlations of H-3 (δH: 3.56 (2H, m)), and H-9 (δH: 4.23 (1H, d, J = 10.5 Hz)) with C-1 (δC: 166.1); H-5 (δH: 2.36 (1H, dd, J = 8.5, 2.5 Hz), 2.10 (1H, m)) and H-8 (δH: 7.66 (1H, s)) with C-6 (δC: 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 δH: 4.08 and H-9 at δH: 4.23, the integration values of H-9 and H-6 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).
Table 3. 1H (500 MHz) and 13C (125 MHz) NMR Date for compound 2 (CDCl3).
Table 3. 1H (500 MHz) and 13C (125 MHz) NMR Date for compound 2 (CDCl3).
No.δH, mult (J in Hz)δCNo.δH, mult (J in Hz)δC
1 166.17 169.7
2 87.66, s
33.56, m45.694.23, d (10.5)53.2
1.89, m22.610α2.32, dd (14.5, 2.0)41.1
2.01, m10β1.79, dd (14.5, 11.0)
2.36, dd (8.5, 2.5)28.311 70.9
2.10, m121.31, s27.4
64.08, t (8.0)58.8131.35, s32.3
Figure 3. The Key HMBC and NOE correlations of compound 2.
Figure 3. The Key HMBC and NOE correlations of compound 2.
Marinedrugs 13 06947 g003
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, 1H, 13C NMR, HSQC, HMBC spectral analysis (Supplementary Materials Figure S27) and comparison of the respective spectral data with those found in the literature. Surprisingly, their 1H and 13C NMR spectral data showed significant differences at positions 1, 2, 3 and 5 (Table 4), though they were tested in the same solvent (DMSO-d6). 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 [17,18,19,20,21,22]. 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 δC: 173.5 in tirandamycin A (3), the data of C-1 in tirandamycin B (4) moved to the downfield at δC: 181.0, implying that 4 should be in 1-keto-4′-enol form. The keto-enol tautomer existed widely 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 δC: 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 δC: 181.0 (C-1), 124.8 (C-2), 143.2 (C-3) and 137.9 (C-5). More importantly, three olefinic protons had obvious differences in these two tautomers in the 1H NMR spectrum, δH: 7.05 (H-2) 7.47 (H-3) and 6.19 (H-5) in 1-enol-4′-keto form changed to δH: 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 δH: 7.55 increased abnormally, which may be due to the shielding effect of the double bond (1C=O). According to the above results and literature survey [17,18,19,20,21,22,24], we also summed up a brief rule that if the 1H NMR data of H-5 is more than δH: 6.00, the structure of tirandamycin is in 1-enol-4′-keto form, otherwise, it is in the other form.
Table 4. The 1H (500 MHz) and 13C (125 MHz) NMR Date for Tirandamycins A and B (DMSO-d6).
Table 4. The 1H (500 MHz) and 13C (125 MHz) NMR Date for Tirandamycins A and B (DMSO-d6).
No.Tirandamycin ATirandamycin B
δH, mult (J in Hz)δCδH, mult (J in Hz)δC
1 173.5 181.0
27.05, d (15.5)116.27.55, d (15.5)124.8
37.47, d (15.5)147.97.14, d (15.5)143.2
4 134.1 134.2
56.19, d (9.5)143.75.81, d (10.0)137.9
62.88, m33.92.83, m33.5
73.77, dd (9.0, 2.0)75.83.73, m76.1
81.81, m33.91.77, m33.7
94.03, d (6.5)77.84.04, d (6.5)77.7
10 202.8 202.5
113.47, s59.93.56, s56.0
12 56.7 59.8
13 96.2 95.3
141.44, s22.41.43, s23.3
151.84, s12.01.79, s12.5
161.07, d (7.0)16.61.05, d (6.5)16.9
170.62, d (7.0)11.00.62, d (6.5)11.1
181.40, s15.13.84, dd (12.5, 3.5) 3.74, dd (12.5, 3.5)56.5
1′8.74, s 7.58, s
2′ 176.0 177.9
3′ 100.7 101.2
4′ 193.9 192.9
5′3.77, dd (9.0, 2.0) 3.75, dd (9.0, 2.0)51.43.47, s50.2
18-OH 5.02 brs
The variable temperature experiments for 1H 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-d6 solution, tirandamycin A (3) was stable in 1-enol-4′-keto form, while 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 data (MS, 1H, 13C 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).
Table 5. Minimum inhibitory concentrations (MIC) for compound 1 and echinomycin.
Table 5. Minimum inhibitory concentrations (MIC) for compound 1 and echinomycin.
MIC (μg/mL)
MicroorganismStaphylococcus epidermidisStaphylococcus aureusEnterococcu faeciumEnterococcus faecalis
Strain No.ATCC 1222812-612-8ATCC 29213ATCC 335911512-2812-33ATCC 70022112-112-3ATCC 29212ATCC 5129912-509-9
Compound 1323232323232323232326416161616
Levofloxacin (conrol)0.250.540.1250.250.1250.2564128128640.511>128
Notes: a Methicillin-susceptible Staphylococcus epidermidis; b Methicillin-resistant Staphylococcus epidermidis; c Methicillin-susceptible Staphylococcus aureus; d Methicillin-resistant Staphylococcus aureus; e Vancomycin-susceptible Enterococcus; f Vancomycin-resistant Enterococcus.
Table 6. Anti-tumor activities (IC50, μM) of compound 1 and echinomycin.
Table 6. Anti-tumor activities (IC50, μM) of compound 1 and echinomycin.
CompoundsIC50 (μM)
ACHNSW1990Mia-PaCa-2786-OU87 MGSK-N-SHJurkatHCT-116NCI-H1650HepG2BGC-823A2780
Compound 10.5522.5604.7500.7210.6275.1740.4148.613.90>10>10>10

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 Technologies, Santa Clara, United States). 1H and 13C NMR, and 2D NMR spectra were obtained at 500 and 125 MHz, using a Bruker AVANCE 500-III spectrometer (Bruker Biospin Group, Karlsruhe, Germany) in chloroform or DMSO-d6 with TMS as an internal reference. HR-ESI-MS data were 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; KNO3 1 g; NaCl 0.5 g; K2HPO3 0.5 g; MgSO4 0.01 g; Natural 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 CH3OH/H2O (20%, 50%, 80%, and 100%, v/v, each 2 L) gradient elution and afforded four primary fractions (A1–A4). Fraction A4 (100% CH3OH, 920 mg) was separated again by Sephadex LH-20 chromatography (2 × 100 cm, 80 g) using 1:10 CHCl3/CH3OH (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% CH3CN in H2O, flow rate 4 mL/min, tR = 37.8 min). Fraction A1 (20% CH3OH/H2O, 715 mg) was separated via Sephadex LH-20 (2 × 100 cm, 80 g) using CH3OH (each 20 mL) as eluent. The fractions were examined by HPLC and the Fraction A1.5 to A1.8 were combined, then it was purified by semipreparative HPLC to provide compound 2 (8.2 mg, 36% CH3CN in H2O, flow rate 4 mL/min, tR = 27.3 min). Fraction A3 (80% CH3OH/H2O, 530 mg) was further separated on RP chromatography (3 × 40 cm, 100 g) eluted with CH3OH/H2O (60%, 70%, 80%, 100%, v/v, each 1 L) and fraction A3.3 (80% CH3OH, 50 mg) was purified by Sephadex LH-20 chromatography (2 × 100 cm, 80 g) eluted with CH3OH to get the yellow band compound 3 (7.5 mg). Fraction A2 (50% CH3OH/H2O, 450 mg) was further separated by semipreparative HPLC to provide compounds 4 (8.0 mg, 42% CH3CN in H2O, flow rate 4 mL/min, tR = 31.5 min)) and 5 (7.8 mg, 47% CH3CN in H2O, flow rate 4 mL/min, tR = 42.6 min).
Compound 1: amorphous yellow powder; [ α ] D 25 −3.8 (c 0.20, CHCl3); UV (CHCl3) λmax (log ε) 245.2 (2.6), 325.8 (1.9); 1H and 13C NMR data, see Table 1; HRESIMS m/z 1101.4288 [M + H]+ (calcd for C51H65N12O12S2, 1101.4286).
Compound 2: white powder; [ α ] D 25 −15.0 (c 0.20, CH3OH); UV (CH3OH) λmax (log ε) 240.2 (3.6); 1H and 13C NMR data, see Table 3; HRESIMS m/z 249.1209 [M + Na]+ (calcd for C11H18N2O3Na, 249.1210).

3.4. Hydrolysis of Compounds 12 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 18 h. After cooling to room temperature, the hydrolysates were dried under reduced pressure and resuspended into 100 μL of H2O.Then they were treated with 1 M NaHCO3 (25 μL), and reacted with 100 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: CH3COOH/H2O, 0.05/99.95, solvent B: CH3CN) 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 H2O (4 μM) were added to 1 M NaHCO3 (20 μL) and 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.

3.5.1. Antibacterial Activity

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), 12-1 (VRE), 12-3 (VSE)), Enterococcus faecalis (ATCC 292121 (VSE), ATCC 51299 (VRE), 12-5 (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 [28,29,30,31].
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 100 μg/mL of streptomycin, in a humidified 5% CO2/air atmosphere at 37 °C. MTT assay: briefly, 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-4-OH-l-Leu) (2), along with three known antibiotics tirandamycin A (3), tirandamycin B (4) and 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.
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.

Supplementary Files

Supplementary File 1


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.


  1. Bhatnagar, I.; Kim, S.-K. Immense essence of excellence: Marine microbial bioactive compounds. Mar. Drugs 2010, 8, 2673–2701. [Google Scholar] [CrossRef] [PubMed]
  2. Blunt, J.W.; Copp, B.R.; Keyzers, R.A.; Munro, M.H.G.; Prinsep, M.R. Marine natural products. Nat. Prod. Rep. 2013, 30, 237–323. [Google Scholar] [CrossRef] [PubMed]
  3. Dharmaraj, S. Marine Streptomyces as a novel source of bioactive substances. World J. Microb. Biot. 2010, 26, 2123–2139. [Google Scholar] [CrossRef]
  4. Balachandran, C.; Duraipandiyan, V.; Emi, N.; Ignacimuthu, S. Antimicrobial and cytotoxic properties of Streptomyces sp.(ERINLG-51) isolated from Southern Western Ghats. South Indian J. Biol. Sci. 2015, 1, 7–14. [Google Scholar]
  5. Sugiyama, R.; Nishimura, S.; Matsumori, N.; Tsunematsu, Y.; Hattori, A.; Kakeya, H. Structure and biological activity of 8-deoxyheronamide C from a marine-derived Streptomyces sp.: Heronamides target saturated hydrocarbon chains in lipid membranes. J. Am. Chem. Soc. 2014, 136, 5209–5212. [Google Scholar] [CrossRef] [PubMed]
  6. Strand, M.; Carlsson, M.; Uvell, H.; Islam, K.; Edlund, K.; Cullman, I.; Altermark, B.; Mei, Y.F.; Elofsson, M.; Willassen, N.P.; et al. Isolation and characterization of anti-adenoviral secondary metabolites from marine actinobacteria. Mar. Drugs 2014, 12, 799–821. [Google Scholar] [CrossRef] [PubMed]
  7. Wang, P.; Kong, F.D.; Wei, J.J.; Wang, Y.; Wang, W.; Hong, K.; Zhu, W.M. Alkaloids from the Mangrove-Derived Actinomycete Jishengella endophytica 161111. Mar. Drugs 2014, 12, 477–490. [Google Scholar] [CrossRef] [PubMed]
  8. Zhang, W.J.; Ma, L.; Li, S.M.; Liu, Z.; Chen, Y.C.; Zhang, H.B.; Zhang, G.T.; Zhang, Q.B.; Tian, X.P.; Yuan, C.S.; et al. Indimicins A–E, Bisindole Alkaloids from the Deep-Sea-Derived Streptomyces sp. SCSIO 03032. J. Nat. Prod. 2014, 77, 1887–1892. [Google Scholar] [CrossRef] [PubMed]
  9. Li, C.Q.; Liu, W.C.; Zhu, P.; Yang, J.L.; Cheng, K.D. Phylogenetic diversity of bacteria associated with the marine sponge Gelliodes carnosa collected from the Hainan Island coastal waters of the South China Sea. Microb. Ecol. 2011, 62, 800–812. [Google Scholar] [CrossRef] [PubMed]
  10. Cui, M.Z.; Gong, T.; Zhu, P. Bioaetive metabolites produced by Streptomyces sp. LS298 isolated from the marine sponge Gelliodes carnosa. Chin. Med. Biotechnol. 2012, 12, 418–425. [Google Scholar]
  11. Wang, Y.; Liu, Y.; Tang, F.; Bernot, K.M.; Schore, R.; Marcucci, G.; Caligiuri, M.A.; Zheng, P.; Liu, Y. Echinomycin protects mice against relapsed acute myeloid leukemia without adverse effect on hematopoietic stem cells. Blood 2014, 124, 1127–1135. [Google Scholar] [CrossRef] [PubMed]
  12. Park, J.Y.; Ryang, Y.S.; Kim, J.B.; Chang, J.-H.; Cho, H.C.; Kim, S.-K. DNA bis-intercalating agent, echinomycin-induced apoptosis via Bcl-2 dependence pathway in human colon cancer cells. Mol. Cell. Toxicol. 2008, 4, 144–149. [Google Scholar]
  13. Park, Y.S.; Shin, W.S.; Kim, S.K. In vitro and in vivo activities of echinomycin against clinical isolates of Staphylococcus aureus. J. Antimicrob. Chemother. 2008, 61, 163–168. [Google Scholar] [CrossRef] [PubMed]
  14. Kim, J.B.; Lee, G.S.; Kim, Y.B.; Kim, S.K.; Kim, Y.H. In vitro antibacterial activity of echinomycin and a novel analogue, YK2000, against vancomycin-resistant enterococci. Int. J. Antimicrob. Agents 2004, 24, 613–615. [Google Scholar] [CrossRef] [PubMed]
  15. Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M.; Harada, K.-I. A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide: Elucidation of Limitations of Marfey’s Method and of Its Separation Mechanism. Anal. Chem. 1997, 69, 3346–3352. [Google Scholar] [CrossRef]
  16. Song, Y.X.; Li, Q.L.; Liu, X.; Chen, Y.C.; Zhang, Y.; Sun, A.J.; Zhang, W.M.; Zhang, J.R.; Ju, J.H. Cyclic Hexapeptides from the Deep South China Sea-Derived Streptomyces scopuliridis SCSIO ZJ46 Active Against Pathogenic Gram-Positive Bacteria. J. Nat. Prod. 2014, 77, 1937–1941. [Google Scholar] [CrossRef] [PubMed]
  17. Lee, V.J.; Rinehart, K.L., Jr. C NMR spectra of streptolydigin, tirandamycin, and related degradation products. J. Antibiot. (Tokyo) 1980, 33, 408–415. [Google Scholar] [CrossRef] [PubMed]
  18. Carlson, J.C.; Li, S.; Burr, D.A.; Sherman, D.H. Isolation and characterization of tirandamycins from a marine-derived Streptomyces sp. J. Nat. Prod. 2009, 72, 2076–2079. [Google Scholar] [CrossRef] [PubMed]
  19. Rateb, M.E.; Yu, Z.G.; Yan, Y.J.; Yang, D.; Huang, T.T.; Vodanovic-Jankovic, S.; Kron, M.A.; Shen, B. Medium optimization of Streptomyces sp. 17944 for tirandamycin B production and isolation and structural elucidation of tirandamycins H, I and J. J. Antibiot. (Tokyo) 2014, 67, 127–132. [Google Scholar] [CrossRef] [PubMed]
  20. Yu, Z.G.; Vodanovic-Jankovic, S.; Ledeboer, N.; Huang, S.X.; Rajski, S.R.; Kron, M.; Shen, B. Tirandamycins from Streptomyces sp. 17944 inhibiting the parasite Brugia malayi asparagine tRNA synthetase. Org. Lett. 2011, 13, 2034–2037. [Google Scholar] [CrossRef] [PubMed]
  21. Duan, C.R.; Yao, Y.L.; Wang, Z.W.; Tian, X.P.; Zhang, S.; Zhang, C. Fermentation optimization, isolation and identification of tirandamycim A and B from marine-derived Streptomyces sp. SCSIO 1666. Chin. J. Mar. Drugs 2010, 29, 12–20. [Google Scholar]
  22. MacKellar, F.A.; Grostic, M.F.; Olson, E.C.; Wnuk, R.J. Tirandamycin. I. Structure assignment. J. Am. Chem. Soc. 1971, 93, 4943–4945. [Google Scholar] [PubMed]
  23. Feng, Z.M.; He, J.; Jiang, J.S.; Chen, Z.; Yang, Y.N.; Zhang, P.C. NMR solution structure study of the representative component hydroxysafflor yellow A and other quinochalcone C-glycosides from Carthamus tinctorius. J. Nat. Prod. 2013, 76, 270–274. [Google Scholar] [CrossRef] [PubMed]
  24. Brill, G.M.; McAlpine, J.B.; Whittern, D. Tirandalydigin, a novel tetramic acid of the tirandamycin-streptolydigin type. II. Isolation and structural characterization. J. Antibiot. (Tokyo) 1988, 41, 36–44. [Google Scholar] [CrossRef] [PubMed]
  25. Takahashi, I.; Asano, K.; Kawamoto, I.; Tamaoki, T.; Nakano, H. UCN-01 and UCN-02, new selective inhibitors of protein kinase C.I. Screening, producing organism and fermentation. J .Antibiot. (Tokyo) 1989, 42, 564–570. [Google Scholar] [CrossRef] [PubMed]
  26. Wikler, M.A. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard, 8th ed.; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2009. [Google Scholar]
  27. Wu, C.Y.; Tan, Y.; Gan, M.L.; Wang, Y.G.; Guan, Y.; Hu, X.X.; Zhou, H.X.; Shang, X.Y.; You, X.F.; Yang, Z.Y.; et al. Identification of elaiophylin derivatives from the marine-derived actinomycete Streptomyces sp. 7-145 using PCR-based screening. J. Nat. Prod. 2013, 76, 2153–2157. [Google Scholar] [CrossRef] [PubMed]
  28. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  29. Carmichael, J.; DeGraff, W.G.; Gazdar, A.F.; Minna, J.D.; Mitchell, J.B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res. 1987, 47, 936–942. [Google Scholar] [PubMed]
  30. He, L.; Liu, Y.M.; Shi, J.G.; Pei, Q. Synthesis and antitumor activity of cholest-4α-methyl-7-en-3β-ol derivatives. Steroids 2006, 71, 476–483. [Google Scholar] [CrossRef] [PubMed]
  31. Gong, T.; Wang, D.X.; Chen, R.Y.; Liu, P.; Yu, D.Q. Novel benzil and isoflavone derivatives from Millettia dielsiana. Planta Med. 2009, 75, 236–242. [Google Scholar] [CrossRef] [PubMed]
  32. Meyer, C.E. Tirandamycin, a new antibiotic isolation and characterization. J. Antibiot. (Tokyo) 1971, 24, 558–560. [Google Scholar] [CrossRef] [PubMed]
Back to TopTop