Letter Cite This: Org. Lett. 2018, 20, 1371−1374
pubs.acs.org/OrgLett
Soliseptide A, A Cyclic Hexapeptide Possessing Piperazic Acid Groups from Streptomyces solisilvae HNM30702 Junfeng Wang,† Ziwen Cong,†,‡ Xiaolong Huang,*,‡ Chenxi Hou,† Weihao Chen,†,∥ Zhengchao Tu,§ Dongyi Huang,‡ and Yonghong Liu*,†,∥ †
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key Laboratory of Marine Materia Medica/RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, People’s Republic of China ‡ Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, People’s Republic of China § Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, People’s Republic of China ∥ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *
ABSTRACT: Soliseptide A (1), a cyclic hexapeptide possessing piperazic acid groups, together with two known azalomycin derivatives (2 and 3) were isolated from Streptomyces solisilvae HNM30702. Their structures were determined through spectroscopic methods and single crystal X-ray diffraction analysis. Soliseptide A (1) possessed a cyclic hexapeptide core featured with two piperazic acid units rarely discovered in nature, and exhibited weak antibacterial and antiviral activities. Besides, compounds 2 and 3 displayed significant fungicidal effects.
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nfectious diseases have seriously imperiled human health worldwide, and antibiotics are important medicines against infectious diseases.1,2 However, the overuse or misuse of the existing antibiotics drugs has triggered widespread resistance and become a global public health problem.3 Microorganisms have attracted attention as new sources for antibiotics drugs. For example, four antibacterial small molecules approved by the U.S. FDA during 2011 to 2014 were either microbial derivednatural products or synthetic derivatives thereof.4 Thus, there is no doubt that it is an irresistible trend to discovery new leading antibiotics with improved activities and/or novel mechanisms of action. As part of our continuing efforts to explore the chemical diversity of microorganisms for drug discovery, an actinomycete strain HNM30702, which was identified as Streptomyces solisilvae, was isolated from the soil sample aroud the rhizosphere of the medicinal plant Cephalotaxus hainanensis and selected for chemical study because its secondary metabolites showed antiphytopathogenic fungi activity against Colletotrichum gloeosporioides. Bioassay-guided fractionation of the culture extracts led to the isolation of one new cyclic hexapeptide soliseptide A (1) (Figure 1), as well as the previously reported two azalomycin derivatives, azalomycins F4a (2) and F5a (3).5,6 Herein, we report the isolation, structural elucidation, antiviral, antibacterial and antifungal activities of these compounds. © 2018 American Chemical Society
Figure 1. Structures of compounds 1−3.
Compound 1 was obtained as colorless needles and crystallized from methanol to give colorless crystals. Its molecular formula was determined to be C31H54N8O9 with 9 degrees of unsaturation from HRESIMS at m/z 683.4089 [M + H]+ (calcd 683.4087) and 705.3932 [M + Na]+ (calcd 705.3906). The relationships between specific proton and carbon signals in the 1H- and 13C NMR data of compound 1 were established by DEPT and HMQC spectra. The presence of six downfield carbon signals for amide or ester carbonyl groups at δC 169.9, 170.9, 171.2, 172.3, 172.9, 174.2, and six αamino acid methine carbons at δC 46.9, 50.4, 53.3, 54.6, 56.3, 59.8 in the 13C NMR spectrum, and four amide proton signals Received: January 13, 2018 Published: February 14, 2018 1371
DOI: 10.1021/acs.orglett.8b00142 Org. Lett. 2018, 20, 1371−1374
Letter
Organic Letters Table 1. 1H and 13C NMR Data for 1 (500, 125 MHz, DMSO-d6, TMS, δ ppm) soliseptide A (1) position Val 1 2 3 4 5 NH Thr 6 7 8 9 NH Pip 10 11 12 13 14 NH
δC 172.3, C 53.3, CH 30.0, CH 19.1, CH3 17.8, CH3
169.9, C 59.8, CH 65.4, CH 20.5, CH3
170.9, C 50.4, CH 26.4, CH2 20.6, CH2 46.9, CH2
δH (J in Hz)
4.86, 1.95, 0.81, 0.80, 7.60,
t (8.0) m d (6.5) d (5.9) d (8.6)
3.92, 3.98, 1.08, 7.74,
t (6.5) m d (6.0) d (5.7)
5.06, 2.06, 1.54, 2.97, 5.43,
brs m; 1.56, m m; 1.46, m m; 2.59, m d (11.0)
position N-Me-Val 15 16 17 18 19 20 4-OH-Pip 21 22 23 24 25 NH N-OH-Leu 26 27 28 29 30 31 N-OH
at δH 7.74, 7.60, 5.49, 5.43 in the 1H NMR spectrum (Table 1), revealed that 1 should be a peptide. Analyses of 1H−1H COSY and HMBC spectra revealed six partial structures, as shown in Figure 2. The COSY correlations of NH (δH 7.60)/H-2/H-3/
δC 171.2, C 56.3, CH 26.6, CH 19.7, CH3 19.4, CH3 30.4, CH3 172.9, C 46.7, CH 30.5, CH2 60.3, CH 52.6, CH2
174.2, C 54.5, CH 36.0, CH2 24.9, CH 23.2, CH3 20.9, CH3
δH (J in Hz)
5.42, 2.15, 0.90, 0.82, 2.99,
brs m d (6.3) d (7.9) s
4.97, 2.03, 3.65, 2.90, 5.49,
d (5.9) m; 1.82, m brs t (13.2); 2.85, t (12.9) d (12.1)
5.68, 1.85, 1.61, 0.90, 0.87, 9.06,
dd (10.9, 2.6) m; 1.37, m m d (6.3) d (6.5) s
Lastly, the COSY correlations of H-27/H2-28/H-29/H3-30(H331) and HMBC correlations of H-27/H2-28 to C-26 (δC 174.2) revealed the presence of an N-substituted leucine moiety. The remaining exchangeable proton (δH 9.06, s) did not show any correlations, suggesting the presence of an N-hydroxyl group, which could match with the molecular formula. This amino acid residue was therefore postulated to be N-hydroxy-leucine (NOH-Leu) (Figures 2 and 3).
Figure 2. Key 1H−1H COSY (bold), and 1H−13C HMBC (arrows) correlations of 1.
H3-5(H3-6) and HMBC correlations of NH (δH 7.60)/H-2/H3 to C-1 (δC 172.3) showed the presence of a valine (Val) moiety. The COSY correlations of NH (δH 7.74)/H-7/H-8/ H3-9, together with that of an α-methine H-7 (δH 3.92) to C-6 (δC 169.9) revealed the presence of a threonine (Thr) moiety. The proximity of an α-methine H-11(δH 5.06) to an NH proton (δH 5.43) and the HMBC correlations between αmethine H-11 (δH 5.06) and C-10 (δC 170.9) suggested the presence of a piperazic acid residue (Pip). The HMBC correlations from dimethyl protons H3-18/H3-19 (δH 0.90/ 0.82) to a methine C-17 (δC 26.6) and α-methine C-16 (δC 56.3), together with those from the α-methine H-16 (δH 5.43) to C-15 (δC 171.2) and from N-methyl group (δH 2.99) to the α-methine C-16 indicated the presence of an N-methyl-valine (N-Me-Val) residue. The 1H−1H COSY spin system from an α-methine H-22 (δH 4.97) to NH (δH 5.49) as well as the oxygenated methine C-24 (δC 60.3) and the HMBC correlation between the α-methine H-22 and C-21 (δC 172.9) suggested the presence of 4-hydroxypiperazic acid (4-OH-Pip) residue.
Figure 3. Gross structure and ORTEP drawing of soliseptide A.
The connectivity between these residues was established by the key HMBC correlations illustrated in Figure 2 as follows: NH (δH 7.60) of Val to C-6 (δC 169.9) of Thr, NH (δH 7.74) of Thr to C-10 (δC 170.9) of Pip, NH (δH 5.43) of Pip to C-15 (δC 171.2) of N-Me-Val, N-CH3 (δH 2.99) of N-Me-Val to C21 (δC 172.9) of 4-OH-Pip. These results revealed that compound (1) is a new cyclic hexapeptide consisting of cyclo(Val-Thr-Pip-(N-Me-Val)-(4-OH-Pip)-(N-OH-Leu)), which was consistent with the 9 degrees of unsaturation from HRESIMS and molecular formula. The suggested sequence were further confirmed by the ESI-MS2 and ESI-MS3 fragment ion series at m/z 665, 564, 353, and 225, corresponding to cleavages of the N−OH bond and amide bonds between Thr/ Pip, Thr/Val, 4-OH-Pip/N-OH-Leu, and 4-OH-Pip/N-Me-Val, 1372
DOI: 10.1021/acs.orglett.8b00142 Org. Lett. 2018, 20, 1371−1374
Letter
Organic Letters Table 2. Inhibitory Effects of Tested Compounds against Pathogenic Bacteria and Fungi pathogenic bacteria and fungi (MIC, μg/mL) compound 1 2 3 ampicillin cycloheximide
Staphylococcus aureus
Streptococcus agalactiae
Colletotrichum gloeosporioides
Colletotrichum asianum
Colletotrichum acutatum
Fusarium oxysporum
Pyricularia oryza
8.0 1.0 2.0 0.125
8.0 2.0 4.0 0.125
>20 1.25 2.5
>20 1.25 5.0
>20 1.25 2.5
>20 2.5 5.0
>20 2.5 5.0
5.0
2.5
2.5
5.0
2.5
respectively (Figure 3). In order to verify the proposed structure including its absolute configuration, compound 1 was subjected to single-crystal X-ray diffraction analysis experiment (CCDC deposition number 1816072) using Cu Kα radiation,7−9 which allowed the assignments of amino acid residues as D-Val, L-Thr, D-Pip, L-N-Me-Val, D-4-OH-Pip, and L-N-OHLeu and gave a 24R configuration for the 4-OH-Pip moiety (Figure 3). Natural products that contain a nitrogen−nitrogen (N−N) bond constitute a fascinating group of compounds with a vast degree of structural diversity, which stimulated many biosynthesis and total synthesis programs.10−12 To date, more than 200 natural products containing an N−N bond have been discovered, of which those possessing the piperazic acid groups number no more than 50.13−15 Among these N−N-containing natural products, molecules containing piperazate (Piz) or its congeners, such as 5-hydroxy-Piz, 5-chloro-Piz, and dehydroPiz, are previously described.13,14 The monamycins were isolated in 1959 by Hassall and co-workers from Streptomyces jamaicensis and represent the first family of natural product that was isolated and contains a piperazic acid motif.16 Soliseptide A (1) possessed a cyclic hexapeptide core featured with two piperazic acid units rarely discovered in nature. Cytotoxicity (TC 50) and anti-EV71 (enterovirus 71) activities (IC50) were determined on Vero cells in vitro using the cytopathic effect (CPE) inhibition assay. Soliseptide A (1) exhibited antiviral activity against EV71 with an IC50 value of 23.0 μM, which was approximately 7-fold more potent than the positive control ribavirin. The selectivity indices (SI) of anti-EV71 activity of soliseptide A (1) was 1.42, which was the ratio of TC50 to IC50 (Supporting Information). Moreover, soliseptide A (1) exhibited weak antibacterial activities against Staphylococcus aureus and Streptococcus agalactiae with MIC values of 8.0 and 8.0 μg/mL, respectively (Table 2). On the basis of the antagonistic behavior (Figure 4d), two antifungal compounds, azalomycins F4a (2) and F5a (3) were also isolated, which showed significant antifungal activities against five phytopathogenic fungi Colletotrichum gloeosporioides, Colletotrichum asianum, Colletotrichum acutatum, Fusarium oxysporum, and Pyricularia oryza, respectively (Figure 4). Besides, azalomycins F4a (2) displayed strong fungicidal effects against above five phytopathogenic fungi, which was stronger than those of the positive control cycloheximide (Table 2). Antifungal activities of azalomycins F4a (2) and F5a (3) indicated that arginine might lead to toxic to these phytopathogenic fungi, which could be regarded as candidate agents of antifungal agrochemicals in the agricultural field.
Figure 4. Colony appearance and micromorphology of strain Streptomyces solisilvae HNM30702 (ISP2). (a) Colony appearance after 7 days at 28 °C. (b) Colony appearance after 20 days at 28 °C. (c) Spiral spore chains as seening using SEM at 28 °C for 2 weeks. Bar, 10.0 μm. (d) Flat confrontation between C. gloeosporioides and HNM30702. (e−i) Antifungal activities at the 10 μg/disc.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00142. The 16S rRNA gene sequences data of Streptomyces solisilvae HNM30702, the NMR, HRESIMS, UV and IR spectra of 1, X-ray crystallographic data of 1, and the NMR, and HRESIMS spectra of 2 and 3 (PDF) Accession Codes
CCDC 1816072 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *Tel/Fax: +86-020-8902-3244. E-mail:
[email protected]. cn. 1373
DOI: 10.1021/acs.orglett.8b00142 Org. Lett. 2018, 20, 1371−1374
Letter
Organic Letters ORCID
Junfeng Wang: 0000-0001-6702-5366 Yonghong Liu: 0000-0001-8327-3108 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 21502204, 21772210, 41776169, and 41476135), Guangdong Province Public Welfare Research and Capacity Building Project (No. 2016A020222010), Pearl River S&T Nova Program of Guangzhou (No. 201710010136), the Strategic Leading Science and Technology Project of CAS (XDA11030403). We are grateful to the analytical facilities in SCSIO.
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DOI: 10.1021/acs.orglett.8b00142 Org. Lett. 2018, 20, 1371−1374
