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Isolation, structure elucidation, and antibacterial evaluation of the metabolites produced by the marine-sourced Streptomyces sp. ZZ820
Tetrahedron 75 (2019) 1186e1193
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Tetrahedron journal homepage: www.elsevier.com/locate/tet
Isolation, structure elucidation, and antibacterial evaluation of the metabolites produced by the marine-sourced Streptomyces sp. ZZ820 Wenwen Yi a, Qiao Li a, Tengfei Song a, Lei Chen a, Xing-Cong Li c, Zhizhen Zhang a, *, Xiao-Yuan Lian b, ** a
Ocean College, Zhoushan Campus, Zhejiang University, Zhoushan 316021, China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2018 Received in revised form 7 January 2019 Accepted 11 January 2019 Available online 14 January 2019
New indole alkaloids streptoprenylindoles AeC (1e3) and diterpenoids 18-acetyl-cyclooctatin (8), 5,18dedihydroxy-cyclooctatin (9), and 5-dehydroxy-cyclooctatin (10) were isolated from the culture of marine-derived Streptomyces sp. ZZ820, along with known 3-cyanomethyl-6-[3-methyl-2-butenyl]indole (4), N-(2-(1H-indol-3-yl)ethylacetamide (5), 1-acetyl-b-carboline (6), indole-3-methylethanoate (7), cyclooctatin (11), and chromomycin A3 (12). Their structures were elucidated by a combination of extensive spectroscopic analyses, ECD calculation, and the Mosher's method. Streptoprenylindoles A (1) and B (2) are enantiomers that were separated through the preparation of their Mosher esters. Three new diterpenoids (8e10) showed antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli with MIC values of 24.11e55.12 mM, while chromomycin A3 (12) showed potent antibacterial activities against MRSA (MIC: 0.59 mM) and E. coli (MIC 0.04 mM). © 2019 Elsevier Ltd. All rights reserved.
Keywords: Streptomyces sp. ZZ820 Marine actinomycetes Streptoprenylindoles Cyclooctatin analogues Antibacterial activities
1. Introduction Marine is a unique environment with extreme variations in terms of nutrients, light, oxygen, pressure, salinity, and temperature. Marine microorganisms have developed remarkable biochemical and physiological capabilities that ensure their survival in this habitat and provide potential for the production of bioactive natural products absent in terrestrial microorganisms [1,2]. Marine actinomycetes are one of the richest producers of bioactive compounds and widely distributed in various marine habitats, such as marine sponges [3], sea sediments [4], mangrove soils and plants [5,6]. It has been reported that the majority of the natural compounds produced by marine actinomycetes are sourced from the single Streptomyces genus (Streptomycetaceae) [1,7,8]. Although representative Streptomyces species are among the most studied actinomycetes, many previously undescribed natural products continued to be discovered from the Streptomyces strains
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X.-Y. Lian).
(Z.
https://doi.org/10.1016/j.tet.2019.01.025 0040-4020/© 2019 Elsevier Ltd. All rights reserved.
Zhang),
[email protected]
in recent years [9e17]. The marine actinomycete Streptomyces sp. ZZ820 was isolated from a sample of coastal soil. A crude extract prepared from the culture of this strain in SGYC liquid medium was found to inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli. Chemical investigation on this bioactive extract resulted in the isolation and identification of 12 compounds including six previously undescribed ones. In this study, we report the isolation and culture of strain ZZ820 as well as the isolation, structure elucidation, and antibacterial activities of the compounds produced by this strain. 2. Results and discussion The strain ZZ820 (Supplementary Data, Fig. S1) was assigned as Streptomyces sp. ZZ820 based on the analysis of its 16S rDNA sequence (Fig. S2), which was similar (99% identity for a 1514 bp stretch of sequence) to the sequence of several Streptomyces strains (Table S1). Large culture of strain ZZ820 in SGYC liquid medium produced compounds 1e12 (Fig. 1). Based on NMR spectroscopic analyses, HRESIMS data, and comparison with reported data, six known compounds were identified as 3-cyanomethyl-6-[3-methyl-
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Fig. 1. Structures of compounds 1e12 isolated from the culture of Streptomyces sp. ZZ820.
2-butenyl]indole (4) [18], N-(2-(1H-indol-3-yl)ethylacetamide (5) [19], 1-acetyl-b-carboline (6) [20,21], indole-3-methylethanoate (7) [22], cyclooctatin (11) [23], and chromomycin A3 (12) [24]. The physical and spectral data of these known compounds are provided in the Supplementary Data. Compounds 1 and 2 were initially isolated as a mixture. In order to separate them, the mixture was treated with the Mosher reagent (S)-a-methoxy-a-(trifluoromethyl) phenylacetyl chloride (S-MTPACl). The resulting reaction mixture was then separated by HPLC to afford R-MTPA ester of 1 and R-MTPA ester of 2 (Fig. S3), which were in turn hydrolyzed by 3 N LiOH to produce pure compounds 1 and 2. Compound 1 was obtained as a white amorphous powder with a positive optical rotation value (þ47.4 ). Its molecular formula C15H18N2O2 was deduced from its HRESIMS ions at m/z [MþH]þ 259.1446 (calcd for C15H19N2O2, 259.1447), [MþNa]þ 281.1262 (calcd for C15H18N2NaO2, 281.1266), and [M H]e 257.1296 (calcd for C15H17N2O2, 257.1290) as well as 13C NMR data. The characteristic NMR signals at dC 124.1 (CH, C-2), 105.0 (C, C-3), 118.7 (CH, C-4), 122.4 (CH, C-5), 135.8 (C, C-6), 113.3 (CH, C-7), 138.7 (C, C-8), 126.1 (C, C-9), and dH 7.17 (1H, s, H-2), 7.49 (1H, d, J ¼ 8.2 Hz, H-4), 7.03 (1H, d, J ¼ 8.2 Hz, H-5), 7.31 (1H, s, H-7) indicated the presence of an indole moiety in 1, which was further supported by the HMBC and 1 H-1H COSY correlations (Fig. 2). A eCH2CN group resonating at dC
Fig. 2. 1H-1H COSY and key HMBC correlations of compounds 1 and 3.
14.4 (CH2, C-10), 120.2 (C, C-11) and dH 3.92 (2H, s, H-10) was linked to C-3 position as evidenced by the HMBC correlations of H-2 with C-10 and H-10 with C-2, C-3, C-9 and C-11. Similarly, the presence of a 2,3-dihydroxy-3-methylbutyl group was confirmed based on its NMR signals at dC 39.3 (CH2, C-12), 81.5 (CH, C-13), 74.0 (C, C-14), 26.3 (CH3, C-15), 24.9 (CH3, C-16), and dH 2.56 (1H, dd, J ¼ 13.9, 10.5 Hz, H-12), 3.10 (1H, dd, J ¼ 13.9, 1.5 Hz, H-12), 3.55 (1H, dd, J ¼ 10.5, 1.5 Hz, H-13), 1.27 (3H, s, H-15), 1.24 (3H, s, H-16), as well as the 1H-1H COSY and HMBC correlations as depicted in Fig. 2. The position of this side chain at C-6 was unambiguously established by a key NOE correlation of H-4 with H-10 and the HMBC correlations of H-4 with C-3, H-5 with C-9, and H-12 with C-5, C-6, and C-7. The absolute configuration at C-13 in compound 1 could be
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determined based on the comparison of its optical rotation value with those of similar compounds, whereby S- and R-configurations correspond to negative and positive optical rotation signs, respectively [25]. Compound 1 was thus assigned as R configuration, which was further confirmed by the Mosher's method as follow. Treatment of 1 with R-MTPA-Cl or S-MTPA-Cl gave its S-MTPA ester (1s) or R-MTPA ester (1r). The 1H NMR chemical shift differences (DdS-R, Fig. 3, Table S5) between 1s and 1r in a positive value for H12 and negative values for both H-15 and H-16 were observed, indicating a 13R-configuration for 1. Therefore, the structure of 1 was determined as 3-cyanomethyl-6-[2(R),3-dihydroxy-3methylbutyl]indole, named as streptoprenylindole A. Its 13C and 1 H NMR data are listed in Tables 1 and 2, respectively. Streptoprenylindole A (1) is structurally close to 3-cyanomethyl-5-[2(S),3dihydroxy-3-methylbutyl]indole, recently isolated from Streptomyces violaceoruber [25]. Their structural difference is the different position of the 2(S),3-dihydroxy-3-methylbutyl group. The molecular formula C15H18N2O2 of compound 2 was confirmed by HRESIMS ions at m/z [MþNa]þ 281.1261 and [MH] 257.1294. The 13C and 1H NMR data (Tables 1 and 2) for 2 and 1 were superimposable, indicating both compounds have the same planer structure. However, compound 2 showed a negative optical rotation value (40.0 ), which was opposite to that of 1, suggesting a 13S-configuration for 2. The configuration at C-13 in 2 was also confirmed by the Mosher's method. The 1H NMR chemical shift differences (DdS-R, Fig. 3, Table S5) between 2s and 2r in a negative value for H-12 and positive values for H-15 and H-16 indicated a 13S-configuration for 2. Therefore, the structure of 2 was determined as 3-cyanomethyl-6-[2(S),3-dihydroxy-3-methylbutyl] indole, named as streptoprenylindole B. The UV, 13C and 1H NMR spectra of 3 suggested that it should be an analogue of 1 or 2. Its HRESIMS spectrum showed an [M H]e ion at m/z 262.1446, corresponding to a molecular formula of C15H21NO3. Comparison of the NMR data (Tables 1 and 2) of 3 with those of 2 indicated that the structural difference at C-3, where a cyanomethyl group (dC 120.2, 14.5; dH 3.92, 2H, s) in 2 was replaced by a hydroxyethyl group (dC 63.9, 30.0; dH 2.93, 3.78, each 2H, t, 7.3 Hz) in 3. Compound 3 should have a 13S-configuration because of its negative optical rotation value. The 13C and 1H assignments of 3 were made by the HSQC and HMBC correlations (Fig. 2). The structure of 3 was thus assigned as 3-hydroxyethyl-6-[2(S),3dihydroxy-3-methylbutyl]indole, named as streptoprenylindole C. Compound 8 was obtained as a colorless oil and had a molecular formula C22H36O4 deduced from HRESIMS ion at m/z [MþNa]þ
Table 1 13 C NMR data for compounds 1e3 (125 MHz, in MeOH-d4) and 8e10 (150 MHz, in DMSO‑d6). No.
1
2
3
8
9
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
e 124.1, CH 105.0, C 118.7, CH 122.4, CH 135.8, C 113.3, CH 138.7, C 126.1, C 14.4, CH2 120.2, C 39.3, CH2 81.5, CH 74.0, C 26.3a, CH3 24.9a, CH3 e e e e e e
e 124.1, CH 105.0, C 118.7, CH 122.4, CH 135.8, C 113.3, CH 138.7, C 126.1, C 14.5, CH2 120.2, C 39.3, CH2 81.6, CH 74.0, C 26.4a, CH3 24.9a, CH3 e e e e e e
e 123.3, CH 112.6, C 119.1, CH 121.5, CH 134.7, C 113.0, CH 138.6, C 127.5, C 30.0, CH2 63.9, CH2 39.4, CH2 81.6, CH 74.0, C 26.3a, CH3 24.9a, CH3 e e e e e e
43.8, CH2 34.2, CH 39.7, CH 38.3, CH2 73.3, CH 56.0, CH 75.9, C 41.2, CH2 118.0, CH 152.1, C 44.2, C 45.0, CH2 22.8, CH2 53.1, CH 28.5, CH 22.0a, CH3 17.2a, CH3 64.4, CH2 26.3, CH3 24.6, CH3 170.4, C 20.7, CH3
45.5, CH2 37.2, CH 39.1, CH 34.2, CH2 26.1, CH2 52.6, CH 74.1, C 41.1, CH2 118.3, CH 151.6, C 44.3, C 45.0, CH2 23.0, CH2 53.1, CH 28.7, CH 22.1a, CH3 17.4a, CH3 15.5, CH3 26.4, CH3 24.7, CH3 e e
44.3, CH2 35.6, CH 47.4, CH 29.4, CH2 26.0, CH2 53.3, CH 73.9, C 41.3, CH2 118.3, CH 151.7, C 44.4, C 44.9, CH2 23.0, CH2 53.1, CH 28.6, CH 22.0a, CH3 17.3a, CH3 61.2, CH2 26.2, CH3 24.7, CH3 e e
a
The data with the same labels in each column may be interchanged.
387.2508. Its 13C NMR and DEPT spectra indicated the presence of one carbonyl (dC 170.4), two olefinic carbons (dC 152.1 and 118.0), one nonprotonated carbon linked to oxygen (dC 75.9), one oxymethine (dC 73.3), one oxymethylene (dC 64.4), one nonprotonated carbon (dC 44.2), five methines (dC 56.0, 53.1, 39.7, 34.2, and 28.5), five methylenes (dC 45.0, 43.8, 41.2, 38.3, and 22.8), and five methyls (dC 26.3, 24.6, 22.0, 20.7, and 17.2). Further analyses of HSQC, 1H-1H COSY, and HMBC correlations (Fig. 4) demonstrated that 8 is an analogue of cyclooctatin (11) [20] with an acetyl moiety at C-18. The absolute configuration at C-5 in 8 was determined by the Mosher's method. Treatment of 8 with R-MTPA-Cl or S-MTPA-Cl gave its SMTPA ester (8s) or R-MTPA ester (8r). The 13C and 1H NMR data (Table S6) of 8s and 8r were assigned by a combination of 1H-1H COSY, HSQC, and HMBC spectroscopic analyses. The 1H NMR chemical shift differences (DdS-R, Fig. 3, Table S6) between 8s and 8r in negative values for H-6, H-8, H-9, and H3-19 and positive values for H-3, H-4, and H3-18 indicated a 5R-configuration. Key NOE correlations (Fig. 4) of H-5 (dH 4.30, m) with H-4b (dH 1.35, m), H-6 (dH 1.82, m), and H3-19 (dH 1.22, s), H-6 with H3-19, H-14 (dH 2.24,
Fig. 3. DdS-R values for the MTPA esters 1s, 1r, 2s, 2r, 8s, and 8r.
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Table 2 1 H NMR data for compounds 1e3 (500 MHz, in MeOH-d4, J in Hz). No.
1
2
3
2 4 5 7 10 11 12
7.17, 1H, s 7.49, 1H, d (8.2) 7.03, 1H, d (8.2) 7.31, 1H, s 3.92, 2H, s e 2.56, 1H, dd (13.9, 10.5); 3.10, 1H, dd (13.9, 1.5) 3.55, 1H, dd (10.5, 1.5) 1.27a, 3H, s 1.24a, 3H, s
7.18, 1H, s 7.49, 1H, d (8.2) 7.03, 1H, dd (8.2, 1.4) 7.30, 1H, s 3.92, 2H, s e 2.56, 1H, dd (13.9, 10.4); 3.10, 1H, dd (13.9, 2.0) 3.55, 1H, dd (10.4, 2.0) 1.27a, 3H, s 1.24a, 3H, s
7.00, 1H, s 7.44, 1H, d (8.1) 6.94, 1H, dd (8.1, 1.3) 7.24, 1H, s 2.93, 2H, t (7.3) 3.78, 2H, t (7.3) 2.54, 1H, dd (13.8, 10.2); 3.07, 1H, dd (13.8, 1.9) 3.55, 1H, dd (10.2, 1.9) 1.26a, 3H, s 1.24a, 3H, s
13 15 16 a
The data with the same labels in each column may be interchanged.
Fig. 4. 1H-1H COSY and key HMBC and NOE correlations of compound 8.
m) with H-12b (dH 1.35, m), and H2-18 with H-1b (dH 1.40, m) and H-4b indicated b-orientation for these protons; while NOE correlations of H-2 (dH 2.42, m) with H3-20 (dH 1.17, s) and with H-1a (dH 1.19, m), H-12a (dH 1.54, m) with H3-20, and H-3 (dH 2.58, m) with H-4a (dH 1.58, m) were suggestive of a-orientation for these protons. A key NOE correlation of H-9 (dH 5.19, m) with H-14 (dH 2.24, m) determined a Z-form for the double bond between C9 and C10. ECD calculation was used to determine the absolute configuration of 8. As shown in Fig. 5, the experimental ECD spectrum of 8 showed a good agreement with the calculated ECD curve of 2R,3R,5R,6S,7S,11R,14R of 8. Based on the foregoing evidence, the structure of 8 was elucidated as 18-acetyl-cyclooctatin, a new diterpenoid. Its 13C and 1H NMR data are shown in Tables 1 and 3. The molecular formula of C20H34O for compound 9 was deduced from its HRESIMS ion at m/z [MþНeН2Ο]þ 273.2576 (calcd for C20H33, 273.2582) and 13C NMR data. Its 13C NMR and DEPT spectra
showed two olefinic carbons (dC 151.6 and 118.3), one nonprotonated carbon linked to oxygen (dC 74.1), one nonprotonated carbon (dC 44.3), five methines (dC 53.1, 52.6, 39.1, 37.2, and 28.7), six methylenes (dC 45.5, 45.0, 41.1, 34.2, 26.1, and 23.0), and five methyls (dC 26.4, 24.7, 22.1, 17.4, and 15.5). Detailed analyses of HSQC, 1H-1H COSY, and HMBC correlations (Fig. 6) suggested that 9 is also an analogue of cyclooctatin (11) without two hydroxyl groups at C-5 and C-18. NOE correlations as shown in Fig. 6 indicated b-orientation for H-6, H-14, H3-18, and H3-19 and a-orientation for H-2, H-3, and H3-20. The absolute configuration of 9 was assigned as 2R,3R,6R,7S,11R,14R based on the result from ECD calculation (Fig. 5). Therefore, the structure of 9 was elucidated as 5,18-dedihydroxycyclooctatin, which is the same compound of cyclooctat-9-en-7-ol, a proposed CotB2 (a diterpenoid cyclase) reaction product [26]. Compound 10 gave an [MþNa]þ ion at m/z 329.2457, 16 mass
Fig. 5. Experimental ECD spectra of compounds 8 and 9 (200e400 nm) in MeOH and the calculated ECD spectra at the B3LYP/6-311 þ g (d, p) level in MeOH.
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Table 3 1 H NMR data for compounds 8e10 (600 MHz, in DMSO‑d6, J in Hz). No.
8
9
10
1
bH: 1.40, m; aH: 1.19, m
bH: 1.47, m; aH: 1.18, m
bH: 1.56, m; aH: 1.16, m
2 3 4
2.42, m 2.58, m bH: 1.35, m; aH: 1.58, m 4.30, m
2.13, m 1.90. m bH: 1.12, m; aH: 1.58, m bH: 1.59, m; aH: 1.48, m 1.77, m 1.82, dd (7.8, 12.1); 2.58, t (12.1) 5.17, m bH: 1.36, m; aH: 1.56, m bH: 1.51, m; aH: 1.31, m 2.24, m 1.76, m 0.91a, d (6.9) 0.74a, d (6.9) 0.81, d (7.1) 0.98, s 1.17, s e 3.97, s e
2.28, m 1.91. m bH: 1.17, m; aH: 1.55, m bH: 1.57, m; aH: 1.48, m 1.81, m 1.83, m; 2.57, t (12.1) 5.18, m bH: 1.34, m; aH: 1.51, m bH: 1.52, m; aH: 1.29, m 2.25, m 1.77, m 0.90a, d (6.9) 0.73a, d (6.9) 3.26, m; 3.40, m 0.96, s 1.16, s e 4.03, s 4.22, t (5.3)
5 6 8 9 12 13 14 15 16 17 18 19 20 21 OH-7 OH-18 a
1.82, m 1.80, m; 2.56, m 5.19, m bH: 1.35, m; aH: 1.54, m bH: 1.52, m; aH: 1.28, m 2.24, m 1.75, m 0.90a, d (7.0) 0.73a, d (7.0) 3.99, m 1.22, s 1.17, s 2.00, s 5.68, s e
The data with the same labels in each column may be interchanged.
Fig. 6. 1H-1H COSY and key HMBC and NOE correlations of compound 9.
units higher than that of 9, implying the presence of an additional oxygen-bearing function in 10. Both 10 and 9 had positive optical rotation values and similar ECD curves. Careful analyses of HSQC, 1 H-1H COSY, HMBC, and NOE correlations (Fig. 7) demonstrated that the structural difference between 10 and 9 is that the methyl (dC 15.5; dH 0.81, 3H, d, J ¼ 7.1 Hz) at C-18 in 9 was substituted by an oxymethylene (dC 61.2; dH 3.26, 3.40, each 1H, m) in 10. Accordingly, the structure of 10 was elucidated as 5-dehydroxycyclooctatin, a
new diterpenoid. The 13C and 1H NMR data of 10 were shown in Tables 1 and 3. Compounds 1e12 were evaluated for their antibacterial activities in inhibiting the growth of MRSA and E. coli by the micro broth dilution method [27]. Gentamicin (an antibiotic against both Grampositive and Gram-negative bacteria) was used as a positive control. The results (Table 4) showed that chromomycin A3 (12) had potent antibacterial activities against MRSA (MIC: 0.59 mM) and
Fig. 7. 1H-1H COSY and key HMBC and NOE correlations of compound 10.
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salt 36 g, tap water 1.0 L) were made in authors' laboratory.
Table 4 Antibacterial activities of compounds 8e12 (MIC: mM). Compounds
8
9
10
11
12
Gentamicin
MRSA E. coli
27.45 35.69
24.11 55.12
29.39 26.12
65.17 58.96
0.59 0.04
0.91 1.82
E. coli (MIC: 0.04 mM). Four diterpenoids 8e11 showed moderate antibacterial activities with MIC values of 24.11e65.17 mM against MRSA and 26.12e58.96 mM against E. coli. The positive control gentamicin gave MIC 0.91 mM against MRSA and 1.82 mM against E. coli. 3. Conclusion Marine-sourced actinomycetes of the Streptomyces genus are one of the richest producers of bioactive natural products. In this study, the culture of marine actinomycete Streptomyces sp. ZZ820 in SGYC liquid medium produced 12 compounds, including previously undescribed streptoprenylindoles AeC (1e3), 18-acetyl-cyclooctatin (8), 5,18-dedihydroxy-cyclooctatin (9), and 5-dehydroxycyclooctatin (10). Structures of the new compounds were determined by extensive spectroscopic analyses, ECD calculation, and the Mosher's method. Streptoprenylindoles A and B (1 and 2) are enantiomers that were separated through the preparation of their Mosher esters. The four cyclooctatin diterpenoids (8e11) showed moderated activities in inhibiting the growth MRSA and E. coli with MIC values of 24.11e65.17 mM and the known chromomycin A3 (12) showed potent antibacterial activities against MRSA (MIC: 0.59 mM) and E. coli (MIC: 0.04 mM), which should be the primary contributor to the antibacterial activities observed for the crude extract.
4.2. Isolation and taxonomic identity of strain ZZ820 Strain ZZ820 was isolated from a soil sample collected from sea coastal located at 30 020 3700 N and 121530 2100 E in East China Sea close to Zhoushan Archipelago (Zhejiang, China) on August 16, 2016. Briefly, the dried soils (1.0 g) were diluted with sterile water to make dilutions of 103, 104, and 105 g/mL. Each dilution (200 mL) was covered on the surface of Gauze's solid medium and then incubated at 28 C for 7 days. The single colony (ZZ820) from the 103 g/mL dilution was picked with sterile needles and transferred to a Gauze's-agar plate. After another 7 days of growth at 28 C, the single colony (ZZ820) that grew well was transferred onto Gauze's agar slants and stored at 4 C for further study. The 16S rDNA analysis of strain ZZ820 was performed by Legenomics (Hangzhou, China) and its DNA sequence using BLAST (nucleotide sequence comparison) was compared to the GenBank database. The 16S rDNA sequence of strain ZZ820 has been deposited in GenBank (accession number: MH388488). The voucher strain of Streptomyces sp. ZZ820 (Streptomycetaceae) was preserved at the Laboratory of Institute of Marine Biology, Ocean College, Zhoushan campus, Zhejiang University, Zhoushan, China. 4.3. Large cultivation of strain ZZ820
4. Experimental section
Colonies of strain ZZ820 from the Gauze's agar plate were inoculated into 500 mL Erlenmeyer flasks, each containing 250 mL of sterile GYM liquid medium and then incubated at 28 C for 3 days on a rotary shaker (180 rpm) to produce seed broth. The seed broth (10 mL) was transferred into a 500 mL Erlenmeyer flask containing 250 mL sterile SGYC medium. All flasks were placed on rotary shakers at 180 rpm for incubation at 28 C for 10 days. A total of 40 L culture was prepared for this study.
4.1. General experimental procedures
4.4. Extraction and isolation of compounds 1e12
UV and IR spectra were recorded on a METASH UV-8000 (Shanghai METASH Instruments Co. Ltd., China) and a Nicolet™ IS™ 10 FT-IR spectrometer (Thermo Fisher Scientific), respectively. ECD spectra were obtained on a JASCO J-815 spectropolarimeter. Optical rotation was measured on a RUDOLPH AutopolⅠAutomatic polarimeter. HRESIMS data were acquired on an Agilent 6230 TOF LC/MS spectrometer. NMR spectra were acquired on a Bruker 500 spectrometer or a JEOL 600 spectrometer using standard programs and acquisition parameters and chemical shifts were expressed in d (ppm). Octadecyl-functionalized silica gel (ODS, Cosmosil 75C18Prep, Nacalai Tesque Inc., Japan), silica gel (100e200, Qingdao Haiyang Chemical Co., Qingdao, China), and Diaion HP-20 (Mitsubishi Chemical, Japan) were used for column chromatography. HPLC separation was performed on a CXTH LC-3000 preparative HPLC system (Beijing Chuangxin tongheng Science & Technology Co. Ltd., China) using a CT-30 column (Fuji-C18, 280 30 mm, 10 mm) or an Agilent 1260 HPLC system using Agilent Zorbax SB-C18 columns (250 9.2 mm, 5 mm, or 250 4.6 mm, 5 mm). Mosher reagents were obtained from Aladdin Industrial Corporation (Shanghai, China). All solvents used for this study were purchased from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 and Escherichia coli ATCC 25922 were gifts from Drs. Zhongjun Ma and Pinmei Wang, respectively. Gentamicin (99.6%) was purchased from Meilune Biotechnology Co. Ltd. (Dalian, China). GYM medium (glucose 4 g, yeast extract 10 g, malt extract 10 g, tap water 1.0 L) and SGYC medium (soluble starch 20 g, glucose 20 g, yeast extract 10 g, corn flour 3 g, KH2PO4 0.5 g, MgSO4$7H2O 0.5 g, CaCO3 2 g, sea
The 40 L culture of strain ZZ820 was centrifuged to yield supernatant and mycelia. The mycelia were extracted with MeOH three times to give mycelium extract. The supernatant was absorbed onto a HP-20 column eluting with water and then MeOH to obtain crude MeOH supernatant extract. The mycelium and supernatant extracts were combined and then partitioned with EtOAc three times to afford a crude extract (4.2 g). This crude extract (4.2 g) was subjected to a column of silica gel eluting with mixed solvents of cyclohexane/EtOAc (10/1, 5/1, 3/1, 2/1, 1/1, v/v), EtOAc, and MeOH to afford seven fractions (Frs. AeG) based on the results of TLC analysis. By using CXTH LC-3000 preparative HPLC (column: Fuji-C18 CT-30, 280 30 mm, 10 mm; flow rate: 10 mL/min; UV detection: 210 nm), compound 9 (58.3 mg, tR 30 min, MeOH/H2O, 98/2) was purified from Fr. A and compound 6 (3.5 mg, tR 30 min, MeOH/H2O, 77/23) from Fr. B. Compound 4 (2 mg, tR 29 min, ACN/ H2O, 63/37) was obtained from Fr. C by Agilent 1260 HPLC purification (column: Agilent Zorbax SB-C18, 250 9.2 mm, 5 mm; flow rate: 1 mL/min; UV detection: 220 nm). Fr. D was fractionated by an ODS column eluting with 80%, 90%, and 100% MeOH to give three subfractions D1eD3. By using the same SB-C18 column and flow rate with a UV detection of 220 nm for 7 and 210 nm for 8, 10, and 11, each of D1eD3 was purified to afford 11 (24.2 mg, tR 21 min, MeOH/ H2O, 90/10), 8 (15 mg, tR 25 min, MeOH/H2O, 90/10), and 10 (21.8 mg, tR 27 min, MeOH/H2O, 90/10), respectively, and compound 7 (4 mg, tR 28 min, MeOH/H2O, 60/40) was obtained from Fr. E. In the same way, Fr. F was separated by an ODS column eluting with 40%, 50%, and 100% MeOH to give three subfractions F1eF3. Further purification of F2 and F3 by the same SB-C18 column and
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flow rate with UV detection of 220 nm gave 3 (4.8 mg, tR 39 min, MeOH/H2O, 47/53) and 5 (1.5 mg, tR 43 min, MeOH/H2O, 47/53) from F2 and a mixture of 1 and 2 (14.2 mg, tR 45 min, MeOH/H2O, 47/53) from F3. Fr. G was also separated by ODS column eluting with 65%, 75%, and 100% MeOH to give G1eG3. Compound 12 (21 mg, tR 29 min, ACN/0.1% HOAc in water, 49/51) was purified from G1 by HPLC using the SB-C18 column (flow rate: 1 mL/min; UV detection: 278 nm). In order to separate compounds 1 and 2, the mixture (14.2 mg) of 1 and 2 was dissolved in 1 mL pyridine and then treated with 90 mL S-MTPA-Cl for 2 h at room temperature. The reaction was terminated by an addition of 0.5 mL MeOH. The resulting mixture was dried in vacuo to give a residue, which was purified by HPLC (column: SB-C18 250 4.6 mm, 5 mm; mobile phase: MeOH/H2O, 60/40; flow rate: 0.8 mL/min; UV detection: 220 nm) to give their R-MTPA esters (1r, tR 34.4 min; 2r, tR 37.2 min). Each of the R-MTPA ester (1r or 2r) was in turn hydrolyzed by LiOH to furnish pure compound 1 or 2. Briefly, compound 1r or 2r (each 6 mg) was dissolved in a mixture of MeOH/H2O (3:1, 2 mL) and then treated with 1 mL 3 N LiOH in MeOH/H2O (3:1) under stirring at room temperature for 12 h. The reaction mixture was neutralized with 3 N HCl and then concentrated under reduced pressure to give a residue, which was purified by HPLC (column: SB-C18 250 4.6 mm, 5 mm; mobile phase: MeOH/H2O, 45/55; flow rate: 0.8 mL/min; UV detection: 220 nm) to give 1 (5.0 mg, tR 11.6 min) or 2 (4.5 mg, tR 11.6 min). 4.4.1. Streptoprenylindole A (1) White amorphous powder; molecular formula C15H18N2O2; [a]20 D þ47.4 (c 0.10, MeOH); UV (MeOH) lmax (log ε) 203 (3.95), 222 (4.17), 274 (3.36), 280 (3.36) nm; IR (MeOH) nmax 3404, 2975, 2931, 2252, 1639, 1454, 1348, 1064 cm1; 13C NMR data (125 MHz, in MeOH-d4), see Table 1, 1H NMR data (500 MHz, in MeOH-d4), see Table 2; HRESIMS m/z [MþH]þ 259.1446 (calcd for C15H19N2O2, 259.1447), [MþNa]þ 281.1262 (calcd for C15H18N2NaO2, 281.1266), and [M H]e 257.1296 (calcd for C15H17N2O2, 257.1290). 4.4.2. Streptoprenylindole B (2) White amorphous powder; molecular formula C15H18N2O2; [a]20 D 40.0 (c 0.10, MeOH); UV (MeOH) lmax (log ε) 203 (3.95), 222 (4.17), 274 (3.36), 280 (3.36) nm; IR (MeOH) nmax 3397, 2977, 2927, 2252, 1627, 1456, 1343, 1066 cm1; 13C NMR data (125 MHz, in MeOH-d4), see Table 1, 1H NMR data (500 MHz, in MeOH-d4), see Table 2; HRESIMS m/z [MþNa]þ 281.1261 (calcd for C15H18N2NaO2, 281.1266) and [M H]e 257.1294 (calcd for C15H17N2O2, 257.1290). 4.4.3. Streptoprenylindole C (3) White amorphous powder; molecular formula C15H21NO3; [a]20 D 18.8 (c 0.10, MeOH); UV (MeOH) lmax (log ε) 205 (4.01), 226 (4.34), 283 (3.54) nm; IR (MeOH) nmax 3306, 2922, 2852, 1454, 1339, 1064, 1029 cm1; 13C NMR data (125 MHz, in MeOH-d4), see Table 1, 1 H NMR data (500 MHz, in MeOH-d4), see Table 2; HRESIMS m/z [M H]e 262.1446 (calcd for C15H20NO3, 262.1443). 4.4.4. 18-Acetyl-cyclooctatin (8) Colorless oil; molecular formula C22H36O4; [a]20 D þ108.6 (c 0.10, MeOH); ECD (9.5 mg/L, MeOH) lmax (Dε) 204 (þ38.5) nm; UV (MeOH) lmax (log ε) 203 (3.93) nm; IR (MeOH) nmax 3315, 2359, 2333, 1738, 1464, 1367, 1249, 1109 cm1; 13C NMR data (150 MHz, in DMSO‑d6), see Table 1, 1H NMR data (600 MHz, in DMSO‑d6), see Table 3; HRESIMS m/z [MþNa]þ 387.2508 (calcd for C22H36NaO4, 387.2511) and [2 MþNa]þ 751.5109 (calcd for C44H72NaO8, 751.5125).
4.4.5. 5,18-Dedihydroxy-cyclooctatin (9) Colorless oil; molecular formula C20H34O; [a]20 D þ105.5 (c 0.10, MeOH); ECD (13.0 mg/L, MeOH) lmax (Dε) 204 (þ27.8) nm; UV (MeOH) lmax (log ε) 203 (3.79) nm; IR (MeOH) nmax 3438, 2361, 2341, 1460, 1375, 1293, 926 cm1; 13C NMR data (150 MHz, in DMSO‑d6), see Table 1, 1H NMR data (600 MHz, in DMSO‑d6), see Table 3; HRESIMS m/z [MþНeН2Ο]þ 273.2576 (calcd for C20H33, 273.2582). 4.4.6. 5-Dehydroxy-cyclooctatin (10) Colorless oil; molecular formula C20H34O2; [a]20 D þ157.5 (c 0.10, MeOH); ECD (10.0 mg/L, MeOH) lmax (Dε) 205 (þ31.5) nm; UV (MeOH) lmax (log ε) 204 (3.94) nm; IR (MeOH) nmax 3434, 2364, 2339, 1458, 1379, 1299, 1109 cm1; 13C NMR data (150 MHz, in DMSO‑d6), see Table 1, 1H NMR data (600 MHz, in DMSO‑d6), see Table 3; HRESIMS m/z [MþNa]þ 329.2457 (calcd for C20H34NaO2, 329.2457). 4.5. MTPA esterification of 1, 2, and 8 A solution of compound 1 (2.0 mg) in 2 mL anhydrous pyridine was divided into two equal portions. R-MTPA-Cl or S-MTPA-Cl (each 45 mL) was added to each portion. Each mixture was stirred for 2 h at room temperature and the reaction was terminated by an addition of 0.5 mL MeOH. Each reaction mixture was concentrated in vacuo to give a residue, which was purified by HPLC (column: SBC18 250 4.6 mm, 5 mm; mobile phase: MeOH/H2O, 60/40; flow rate: 0.8 mL/min; UV detection: 220 nm) to give 1s (S-MTPA ester of 1, 0.5 mg, tR 37.2 min) or 1r {R-MTPA ester of 1, 0.7 mg, tR 34.4 min, HRESIMS m/z [MþNa]þ 497.1661 (calcd for C25H25F3N2NaO4, 497.1664)} for NMR analysis. Compound 2 (2.0 mg) was also treated by R-MTPA-Cl or S-MTPA-Cl to afford 2s (S-MTPA ester of 2, 0.7 mg, tR 34.4 min) or 2r {R-MTPA ester of 2, 0.5 mg, tR 37.2 min, HRESIMS m/z [MþNa]þ 497.1660 (calcd for C25H25F3N2NaO4, 497.1664)} for NMR analysis. The 1H NMR data for 1s, 1r, 2s, and 2r are summarized in Table S5. In the same way, compound 8 (2.0 mg) was reacted with R-MTPA-Cl or S-MTPA-Cl to afford 8s (S-MTPA ester of 8, 1.8 mg, tR 24.7 min, MeOH/H2O, 95/5; flow rate, 1.0 mL/min) or 8r (R-MTPA ester of 8, 1.9 mg, tR 23.6 min) for NMR analysis. The 13C and 1H NMR data for 8s and 8r are summarized in Table S6. 4.6. ECD calculation Monte Carlo conformational searches were carried out by means of the Spartan's 10 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmann-population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31 þ g (d, p) level in MeOH using the CPCM polarizable conductor calculation model (Figs. S131 and S132 and Tables S7 and S9). The theoretical calculation of ECD was conducted in MeOH using Time-dependent Density functional theory (TD-DFT) at the B3LYP/6-311 þ g (d, p) level for all conformers of compounds 8 and 9 (Tables S8 and S10). Rotatory strengths for a total of 50 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma ¼ 0.3 eV. 4.7. Antibacterial assay The antibacterial activities of compounds 1e12 against the growth of MRSA and E. coli were determined by the micro broth dilution method as described in previous publication [27]. Gentamicin was used as a positive control and DMSO was used as a
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negative control. Conflicts of interest The authors declare no conflict of interest. Acknowledgments This study was supported by the National Key R&D Program of China (No. 2017YFE0102200) and the National Natural Science Foundation of China (81773587, 81773769, and 81273428). We appreciate Dr. Jianyang Pan (Pharmaceutical Informatics Institute of Zhejiang University) for performing the NMR spectrometry. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.tet.2019.01.025. References [1] M. Kamjam, P. Sivalingam, Z. Deng, K. Hong, Deep sea actinomycetes and their secondary metabolites, Front. Microbiol. 8 (2017) 760. [2] C. Zhao, T. Zhu, W. Zhu, Chin. J. Org. Chem. 33 (2013) 1195e1234. [3] U.R. Abdelmohsen, K. Bayer, U. Hentschel, Nat. Prod. Rep. 31 (2014) 381e399. [4] P. Chen, L. Zhang, X. Guo, X. Dai, L. Liu, L. Xi, J. Wang, L. Song, Y. Wang, Y. Zhu, L. Huang, Y. Huang, Front. Microbiol. 7 (2016) 1340. [5] A.S. Azman, I. Othman, S.S. Velu, K.G. Chan, L.H. Lee, Front. Microbiol. 6 (2015) 856. [6] K. Hong, A.H. Gao, Q.Y. Xie, H. Gao, L. Zhuang, H.P. Lin, H.P. Yu, J. Li, X.S. Yao, M. Goodfellow, J.S. Ruan, Mar. Drugs 7 (2009) 24e44. [7] P.G. Williams, Trends Biotechnol. 27 (2009) 45e52. [8] P. Manivasagan, J. Venkatesan, K. Sivakumar, S.K. Kim, Microbiol. Res. 169
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(2014) 262e278. [9] P. Fu, C. Yang, Y. Wang, P. Liu, Y. Ma, L. Xu, M. Su, K. Hong, W. Zhu, Org. Lett. 14 (2012) 2422e2425. [10] C. Wang, X. Mei, W. Zhu, Stud. Mar. Sin. 51 (2016) 86e124. [11] M. Bae, K. Moon, J. Kim, H.J. Park, S.K. Lee, J. Shin, D.C. Oh, J. Nat. Prod. 79 (2016) 332e339. [12] L. Chen, W. Chai, W. Wang, T. Song, X.Y. Lian, Z. Zhang, J. Nat. Prod. 80 (2017) 1450e1456. [13] W. Wang, T. Song, W. Chai, L. Chen, L. Chen, X.Y. Lian, Z. Zhang, Sci. Rep. 7 (2017) 1703. [14] X. Ye, K. Anjum, T. Song, W. Wang, Y. Liang, M. Chen, H. Huang, X.Y. Lian, Z. Zhang, Phytochemistry 135 (2017) 151e159. [15] X. Zhang, L. Chen, W. Chai, X.Y. Lian, Z. Zhang, Phytochemistry 44 (2017) 119e126. [16] M. Chen, W. Chai, T. Song, M. Ma, X.Y. Lian, Z. Zhang, Sci. Rep. 8 (2018) 72. [17] M. Chen, W. Chai, R. Zhu, T. Song, Z. Zhang, X.Y. Lian, Tetrahedron 74 (2018) 2100e2106. pez, M. Martínez Insua, J. Pe rez Baz, J.L. Ferna ndez Puentes, [18] J.M. S anchez Lo ~ edo Herna ndez, J. Nat. Prod. 66 (2003) 863e864. L.M. Can , D. Stien, B.K. Amian, P.A. Blandinie res, [19] J.B.S. Anouhe, A.A. Adima, F.B. Niamke D. Virieux, J.L. Pirat, S. Kati-Coulibaly, N. Amusant, Phytochem. Lett. 12 (2015) 158e163. [20] H. Huang, Y. Yao, Z. He, T. Yang, J. Ma, X. Tian, Y. Li, C. Huang, X. Chen, W. Li, S. Zhang, C. Zhang, J. Ju, J. Nat. Prod. 74 (2011) 2122e2127. [21] X.J. Zhao, H.F. Wang, D.Q. Zhao, H.X. Ji, Y.H. Pei, J. Diels Bai, J. Shenyang Pharm. Uni. 30 (2013) 182e185. [22] Y. Liu, J.H. Jung, S. Zhang, Syst. Ecol. 34 (2006) 453e456. [23] A. Kawamura, M. Iacovidou, E. Hirokawa, C.E. Soll, M. Trujillo, J. Nat. Prod. 74 (2011) 492e495. [24] K. Toume, K. Tsukahara, H. Ito, M.A. Arai, M. Ishibashi, Mar. Drugs 12 (2014) 3466e3476. [25] J. Ma, H. Lei, X. Chen, X. Bi, Y. Jiang, L. Han, X. Huang, J. Antibiot. 70 (2017) 991e994. [26] A. Meguro, Y. Motoyoshi, K. Teramoto, S. Ueda, Y. Totsuka, Y. Ando, T. Tomita, S.Y. Kim, T. Kimura, M. Igarashi, R. Sawa, T. Shinada, M. Nishiyama, T. Kuzuyama, Angew Chem. Int. Ed. Engl. 54 (2015) 4353e4356. [27] X. Ye, K. Anjum, T. Song, W. Wang, S. Yu, H. Huang, X.Y. Lian, Z. Zhang, Nat. Prod. Res. 30 (2016) 1156e1161.
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Isolation, structure elucidation, and antibacterial evaluation of the metabolites produced by the marine-sourced Streptomyces sp. ZZ820 Wenwen Yi a, Qiao Li a, Tengfei Song a, Lei Chen a, Xing-Cong Li c, Zhizhen Zhang a, *, Xiao-Yuan Lian b, ** a
Ocean College, Zhoushan Campus, Zhejiang University, Zhoushan 316021, China College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2018 Received in revised form 7 January 2019 Accepted 11 January 2019 Available online 14 January 2019
New indole alkaloids streptoprenylindoles AeC (1e3) and diterpenoids 18-acetyl-cyclooctatin (8), 5,18dedihydroxy-cyclooctatin (9), and 5-dehydroxy-cyclooctatin (10) were isolated from the culture of marine-derived Streptomyces sp. ZZ820, along with known 3-cyanomethyl-6-[3-methyl-2-butenyl]indole (4), N-(2-(1H-indol-3-yl)ethylacetamide (5), 1-acetyl-b-carboline (6), indole-3-methylethanoate (7), cyclooctatin (11), and chromomycin A3 (12). Their structures were elucidated by a combination of extensive spectroscopic analyses, ECD calculation, and the Mosher's method. Streptoprenylindoles A (1) and B (2) are enantiomers that were separated through the preparation of their Mosher esters. Three new diterpenoids (8e10) showed antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli with MIC values of 24.11e55.12 mM, while chromomycin A3 (12) showed potent antibacterial activities against MRSA (MIC: 0.59 mM) and E. coli (MIC 0.04 mM). © 2019 Elsevier Ltd. All rights reserved.
Keywords: Streptomyces sp. ZZ820 Marine actinomycetes Streptoprenylindoles Cyclooctatin analogues Antibacterial activities
1. Introduction Marine is a unique environment with extreme variations in terms of nutrients, light, oxygen, pressure, salinity, and temperature. Marine microorganisms have developed remarkable biochemical and physiological capabilities that ensure their survival in this habitat and provide potential for the production of bioactive natural products absent in terrestrial microorganisms [1,2]. Marine actinomycetes are one of the richest producers of bioactive compounds and widely distributed in various marine habitats, such as marine sponges [3], sea sediments [4], mangrove soils and plants [5,6]. It has been reported that the majority of the natural compounds produced by marine actinomycetes are sourced from the single Streptomyces genus (Streptomycetaceae) [1,7,8]. Although representative Streptomyces species are among the most studied actinomycetes, many previously undescribed natural products continued to be discovered from the Streptomyces strains
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (X.-Y. Lian).
(Z.
https://doi.org/10.1016/j.tet.2019.01.025 0040-4020/© 2019 Elsevier Ltd. All rights reserved.
Zhang),
[email protected]
in recent years [9e17]. The marine actinomycete Streptomyces sp. ZZ820 was isolated from a sample of coastal soil. A crude extract prepared from the culture of this strain in SGYC liquid medium was found to inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli. Chemical investigation on this bioactive extract resulted in the isolation and identification of 12 compounds including six previously undescribed ones. In this study, we report the isolation and culture of strain ZZ820 as well as the isolation, structure elucidation, and antibacterial activities of the compounds produced by this strain. 2. Results and discussion The strain ZZ820 (Supplementary Data, Fig. S1) was assigned as Streptomyces sp. ZZ820 based on the analysis of its 16S rDNA sequence (Fig. S2), which was similar (99% identity for a 1514 bp stretch of sequence) to the sequence of several Streptomyces strains (Table S1). Large culture of strain ZZ820 in SGYC liquid medium produced compounds 1e12 (Fig. 1). Based on NMR spectroscopic analyses, HRESIMS data, and comparison with reported data, six known compounds were identified as 3-cyanomethyl-6-[3-methyl-
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Fig. 1. Structures of compounds 1e12 isolated from the culture of Streptomyces sp. ZZ820.
2-butenyl]indole (4) [18], N-(2-(1H-indol-3-yl)ethylacetamide (5) [19], 1-acetyl-b-carboline (6) [20,21], indole-3-methylethanoate (7) [22], cyclooctatin (11) [23], and chromomycin A3 (12) [24]. The physical and spectral data of these known compounds are provided in the Supplementary Data. Compounds 1 and 2 were initially isolated as a mixture. In order to separate them, the mixture was treated with the Mosher reagent (S)-a-methoxy-a-(trifluoromethyl) phenylacetyl chloride (S-MTPACl). The resulting reaction mixture was then separated by HPLC to afford R-MTPA ester of 1 and R-MTPA ester of 2 (Fig. S3), which were in turn hydrolyzed by 3 N LiOH to produce pure compounds 1 and 2. Compound 1 was obtained as a white amorphous powder with a positive optical rotation value (þ47.4 ). Its molecular formula C15H18N2O2 was deduced from its HRESIMS ions at m/z [MþH]þ 259.1446 (calcd for C15H19N2O2, 259.1447), [MþNa]þ 281.1262 (calcd for C15H18N2NaO2, 281.1266), and [M H]e 257.1296 (calcd for C15H17N2O2, 257.1290) as well as 13C NMR data. The characteristic NMR signals at dC 124.1 (CH, C-2), 105.0 (C, C-3), 118.7 (CH, C-4), 122.4 (CH, C-5), 135.8 (C, C-6), 113.3 (CH, C-7), 138.7 (C, C-8), 126.1 (C, C-9), and dH 7.17 (1H, s, H-2), 7.49 (1H, d, J ¼ 8.2 Hz, H-4), 7.03 (1H, d, J ¼ 8.2 Hz, H-5), 7.31 (1H, s, H-7) indicated the presence of an indole moiety in 1, which was further supported by the HMBC and 1 H-1H COSY correlations (Fig. 2). A eCH2CN group resonating at dC
Fig. 2. 1H-1H COSY and key HMBC correlations of compounds 1 and 3.
14.4 (CH2, C-10), 120.2 (C, C-11) and dH 3.92 (2H, s, H-10) was linked to C-3 position as evidenced by the HMBC correlations of H-2 with C-10 and H-10 with C-2, C-3, C-9 and C-11. Similarly, the presence of a 2,3-dihydroxy-3-methylbutyl group was confirmed based on its NMR signals at dC 39.3 (CH2, C-12), 81.5 (CH, C-13), 74.0 (C, C-14), 26.3 (CH3, C-15), 24.9 (CH3, C-16), and dH 2.56 (1H, dd, J ¼ 13.9, 10.5 Hz, H-12), 3.10 (1H, dd, J ¼ 13.9, 1.5 Hz, H-12), 3.55 (1H, dd, J ¼ 10.5, 1.5 Hz, H-13), 1.27 (3H, s, H-15), 1.24 (3H, s, H-16), as well as the 1H-1H COSY and HMBC correlations as depicted in Fig. 2. The position of this side chain at C-6 was unambiguously established by a key NOE correlation of H-4 with H-10 and the HMBC correlations of H-4 with C-3, H-5 with C-9, and H-12 with C-5, C-6, and C-7. The absolute configuration at C-13 in compound 1 could be
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determined based on the comparison of its optical rotation value with those of similar compounds, whereby S- and R-configurations correspond to negative and positive optical rotation signs, respectively [25]. Compound 1 was thus assigned as R configuration, which was further confirmed by the Mosher's method as follow. Treatment of 1 with R-MTPA-Cl or S-MTPA-Cl gave its S-MTPA ester (1s) or R-MTPA ester (1r). The 1H NMR chemical shift differences (DdS-R, Fig. 3, Table S5) between 1s and 1r in a positive value for H12 and negative values for both H-15 and H-16 were observed, indicating a 13R-configuration for 1. Therefore, the structure of 1 was determined as 3-cyanomethyl-6-[2(R),3-dihydroxy-3methylbutyl]indole, named as streptoprenylindole A. Its 13C and 1 H NMR data are listed in Tables 1 and 2, respectively. Streptoprenylindole A (1) is structurally close to 3-cyanomethyl-5-[2(S),3dihydroxy-3-methylbutyl]indole, recently isolated from Streptomyces violaceoruber [25]. Their structural difference is the different position of the 2(S),3-dihydroxy-3-methylbutyl group. The molecular formula C15H18N2O2 of compound 2 was confirmed by HRESIMS ions at m/z [MþNa]þ 281.1261 and [MH] 257.1294. The 13C and 1H NMR data (Tables 1 and 2) for 2 and 1 were superimposable, indicating both compounds have the same planer structure. However, compound 2 showed a negative optical rotation value (40.0 ), which was opposite to that of 1, suggesting a 13S-configuration for 2. The configuration at C-13 in 2 was also confirmed by the Mosher's method. The 1H NMR chemical shift differences (DdS-R, Fig. 3, Table S5) between 2s and 2r in a negative value for H-12 and positive values for H-15 and H-16 indicated a 13S-configuration for 2. Therefore, the structure of 2 was determined as 3-cyanomethyl-6-[2(S),3-dihydroxy-3-methylbutyl] indole, named as streptoprenylindole B. The UV, 13C and 1H NMR spectra of 3 suggested that it should be an analogue of 1 or 2. Its HRESIMS spectrum showed an [M H]e ion at m/z 262.1446, corresponding to a molecular formula of C15H21NO3. Comparison of the NMR data (Tables 1 and 2) of 3 with those of 2 indicated that the structural difference at C-3, where a cyanomethyl group (dC 120.2, 14.5; dH 3.92, 2H, s) in 2 was replaced by a hydroxyethyl group (dC 63.9, 30.0; dH 2.93, 3.78, each 2H, t, 7.3 Hz) in 3. Compound 3 should have a 13S-configuration because of its negative optical rotation value. The 13C and 1H assignments of 3 were made by the HSQC and HMBC correlations (Fig. 2). The structure of 3 was thus assigned as 3-hydroxyethyl-6-[2(S),3dihydroxy-3-methylbutyl]indole, named as streptoprenylindole C. Compound 8 was obtained as a colorless oil and had a molecular formula C22H36O4 deduced from HRESIMS ion at m/z [MþNa]þ
Table 1 13 C NMR data for compounds 1e3 (125 MHz, in MeOH-d4) and 8e10 (150 MHz, in DMSO‑d6). No.
1
2
3
8
9
10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
e 124.1, CH 105.0, C 118.7, CH 122.4, CH 135.8, C 113.3, CH 138.7, C 126.1, C 14.4, CH2 120.2, C 39.3, CH2 81.5, CH 74.0, C 26.3a, CH3 24.9a, CH3 e e e e e e
e 124.1, CH 105.0, C 118.7, CH 122.4, CH 135.8, C 113.3, CH 138.7, C 126.1, C 14.5, CH2 120.2, C 39.3, CH2 81.6, CH 74.0, C 26.4a, CH3 24.9a, CH3 e e e e e e
e 123.3, CH 112.6, C 119.1, CH 121.5, CH 134.7, C 113.0, CH 138.6, C 127.5, C 30.0, CH2 63.9, CH2 39.4, CH2 81.6, CH 74.0, C 26.3a, CH3 24.9a, CH3 e e e e e e
43.8, CH2 34.2, CH 39.7, CH 38.3, CH2 73.3, CH 56.0, CH 75.9, C 41.2, CH2 118.0, CH 152.1, C 44.2, C 45.0, CH2 22.8, CH2 53.1, CH 28.5, CH 22.0a, CH3 17.2a, CH3 64.4, CH2 26.3, CH3 24.6, CH3 170.4, C 20.7, CH3
45.5, CH2 37.2, CH 39.1, CH 34.2, CH2 26.1, CH2 52.6, CH 74.1, C 41.1, CH2 118.3, CH 151.6, C 44.3, C 45.0, CH2 23.0, CH2 53.1, CH 28.7, CH 22.1a, CH3 17.4a, CH3 15.5, CH3 26.4, CH3 24.7, CH3 e e
44.3, CH2 35.6, CH 47.4, CH 29.4, CH2 26.0, CH2 53.3, CH 73.9, C 41.3, CH2 118.3, CH 151.7, C 44.4, C 44.9, CH2 23.0, CH2 53.1, CH 28.6, CH 22.0a, CH3 17.3a, CH3 61.2, CH2 26.2, CH3 24.7, CH3 e e
a
The data with the same labels in each column may be interchanged.
387.2508. Its 13C NMR and DEPT spectra indicated the presence of one carbonyl (dC 170.4), two olefinic carbons (dC 152.1 and 118.0), one nonprotonated carbon linked to oxygen (dC 75.9), one oxymethine (dC 73.3), one oxymethylene (dC 64.4), one nonprotonated carbon (dC 44.2), five methines (dC 56.0, 53.1, 39.7, 34.2, and 28.5), five methylenes (dC 45.0, 43.8, 41.2, 38.3, and 22.8), and five methyls (dC 26.3, 24.6, 22.0, 20.7, and 17.2). Further analyses of HSQC, 1H-1H COSY, and HMBC correlations (Fig. 4) demonstrated that 8 is an analogue of cyclooctatin (11) [20] with an acetyl moiety at C-18. The absolute configuration at C-5 in 8 was determined by the Mosher's method. Treatment of 8 with R-MTPA-Cl or S-MTPA-Cl gave its SMTPA ester (8s) or R-MTPA ester (8r). The 13C and 1H NMR data (Table S6) of 8s and 8r were assigned by a combination of 1H-1H COSY, HSQC, and HMBC spectroscopic analyses. The 1H NMR chemical shift differences (DdS-R, Fig. 3, Table S6) between 8s and 8r in negative values for H-6, H-8, H-9, and H3-19 and positive values for H-3, H-4, and H3-18 indicated a 5R-configuration. Key NOE correlations (Fig. 4) of H-5 (dH 4.30, m) with H-4b (dH 1.35, m), H-6 (dH 1.82, m), and H3-19 (dH 1.22, s), H-6 with H3-19, H-14 (dH 2.24,
Fig. 3. DdS-R values for the MTPA esters 1s, 1r, 2s, 2r, 8s, and 8r.
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Table 2 1 H NMR data for compounds 1e3 (500 MHz, in MeOH-d4, J in Hz). No.
1
2
3
2 4 5 7 10 11 12
7.17, 1H, s 7.49, 1H, d (8.2) 7.03, 1H, d (8.2) 7.31, 1H, s 3.92, 2H, s e 2.56, 1H, dd (13.9, 10.5); 3.10, 1H, dd (13.9, 1.5) 3.55, 1H, dd (10.5, 1.5) 1.27a, 3H, s 1.24a, 3H, s
7.18, 1H, s 7.49, 1H, d (8.2) 7.03, 1H, dd (8.2, 1.4) 7.30, 1H, s 3.92, 2H, s e 2.56, 1H, dd (13.9, 10.4); 3.10, 1H, dd (13.9, 2.0) 3.55, 1H, dd (10.4, 2.0) 1.27a, 3H, s 1.24a, 3H, s
7.00, 1H, s 7.44, 1H, d (8.1) 6.94, 1H, dd (8.1, 1.3) 7.24, 1H, s 2.93, 2H, t (7.3) 3.78, 2H, t (7.3) 2.54, 1H, dd (13.8, 10.2); 3.07, 1H, dd (13.8, 1.9) 3.55, 1H, dd (10.2, 1.9) 1.26a, 3H, s 1.24a, 3H, s
13 15 16 a
The data with the same labels in each column may be interchanged.
Fig. 4. 1H-1H COSY and key HMBC and NOE correlations of compound 8.
m) with H-12b (dH 1.35, m), and H2-18 with H-1b (dH 1.40, m) and H-4b indicated b-orientation for these protons; while NOE correlations of H-2 (dH 2.42, m) with H3-20 (dH 1.17, s) and with H-1a (dH 1.19, m), H-12a (dH 1.54, m) with H3-20, and H-3 (dH 2.58, m) with H-4a (dH 1.58, m) were suggestive of a-orientation for these protons. A key NOE correlation of H-9 (dH 5.19, m) with H-14 (dH 2.24, m) determined a Z-form for the double bond between C9 and C10. ECD calculation was used to determine the absolute configuration of 8. As shown in Fig. 5, the experimental ECD spectrum of 8 showed a good agreement with the calculated ECD curve of 2R,3R,5R,6S,7S,11R,14R of 8. Based on the foregoing evidence, the structure of 8 was elucidated as 18-acetyl-cyclooctatin, a new diterpenoid. Its 13C and 1H NMR data are shown in Tables 1 and 3. The molecular formula of C20H34O for compound 9 was deduced from its HRESIMS ion at m/z [MþНeН2Ο]þ 273.2576 (calcd for C20H33, 273.2582) and 13C NMR data. Its 13C NMR and DEPT spectra
showed two olefinic carbons (dC 151.6 and 118.3), one nonprotonated carbon linked to oxygen (dC 74.1), one nonprotonated carbon (dC 44.3), five methines (dC 53.1, 52.6, 39.1, 37.2, and 28.7), six methylenes (dC 45.5, 45.0, 41.1, 34.2, 26.1, and 23.0), and five methyls (dC 26.4, 24.7, 22.1, 17.4, and 15.5). Detailed analyses of HSQC, 1H-1H COSY, and HMBC correlations (Fig. 6) suggested that 9 is also an analogue of cyclooctatin (11) without two hydroxyl groups at C-5 and C-18. NOE correlations as shown in Fig. 6 indicated b-orientation for H-6, H-14, H3-18, and H3-19 and a-orientation for H-2, H-3, and H3-20. The absolute configuration of 9 was assigned as 2R,3R,6R,7S,11R,14R based on the result from ECD calculation (Fig. 5). Therefore, the structure of 9 was elucidated as 5,18-dedihydroxycyclooctatin, which is the same compound of cyclooctat-9-en-7-ol, a proposed CotB2 (a diterpenoid cyclase) reaction product [26]. Compound 10 gave an [MþNa]þ ion at m/z 329.2457, 16 mass
Fig. 5. Experimental ECD spectra of compounds 8 and 9 (200e400 nm) in MeOH and the calculated ECD spectra at the B3LYP/6-311 þ g (d, p) level in MeOH.
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Table 3 1 H NMR data for compounds 8e10 (600 MHz, in DMSO‑d6, J in Hz). No.
8
9
10
1
bH: 1.40, m; aH: 1.19, m
bH: 1.47, m; aH: 1.18, m
bH: 1.56, m; aH: 1.16, m
2 3 4
2.42, m 2.58, m bH: 1.35, m; aH: 1.58, m 4.30, m
2.13, m 1.90. m bH: 1.12, m; aH: 1.58, m bH: 1.59, m; aH: 1.48, m 1.77, m 1.82, dd (7.8, 12.1); 2.58, t (12.1) 5.17, m bH: 1.36, m; aH: 1.56, m bH: 1.51, m; aH: 1.31, m 2.24, m 1.76, m 0.91a, d (6.9) 0.74a, d (6.9) 0.81, d (7.1) 0.98, s 1.17, s e 3.97, s e
2.28, m 1.91. m bH: 1.17, m; aH: 1.55, m bH: 1.57, m; aH: 1.48, m 1.81, m 1.83, m; 2.57, t (12.1) 5.18, m bH: 1.34, m; aH: 1.51, m bH: 1.52, m; aH: 1.29, m 2.25, m 1.77, m 0.90a, d (6.9) 0.73a, d (6.9) 3.26, m; 3.40, m 0.96, s 1.16, s e 4.03, s 4.22, t (5.3)
5 6 8 9 12 13 14 15 16 17 18 19 20 21 OH-7 OH-18 a
1.82, m 1.80, m; 2.56, m 5.19, m bH: 1.35, m; aH: 1.54, m bH: 1.52, m; aH: 1.28, m 2.24, m 1.75, m 0.90a, d (7.0) 0.73a, d (7.0) 3.99, m 1.22, s 1.17, s 2.00, s 5.68, s e
The data with the same labels in each column may be interchanged.
Fig. 6. 1H-1H COSY and key HMBC and NOE correlations of compound 9.
units higher than that of 9, implying the presence of an additional oxygen-bearing function in 10. Both 10 and 9 had positive optical rotation values and similar ECD curves. Careful analyses of HSQC, 1 H-1H COSY, HMBC, and NOE correlations (Fig. 7) demonstrated that the structural difference between 10 and 9 is that the methyl (dC 15.5; dH 0.81, 3H, d, J ¼ 7.1 Hz) at C-18 in 9 was substituted by an oxymethylene (dC 61.2; dH 3.26, 3.40, each 1H, m) in 10. Accordingly, the structure of 10 was elucidated as 5-dehydroxycyclooctatin, a
new diterpenoid. The 13C and 1H NMR data of 10 were shown in Tables 1 and 3. Compounds 1e12 were evaluated for their antibacterial activities in inhibiting the growth of MRSA and E. coli by the micro broth dilution method [27]. Gentamicin (an antibiotic against both Grampositive and Gram-negative bacteria) was used as a positive control. The results (Table 4) showed that chromomycin A3 (12) had potent antibacterial activities against MRSA (MIC: 0.59 mM) and
Fig. 7. 1H-1H COSY and key HMBC and NOE correlations of compound 10.
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salt 36 g, tap water 1.0 L) were made in authors' laboratory.
Table 4 Antibacterial activities of compounds 8e12 (MIC: mM). Compounds
8
9
10
11
12
Gentamicin
MRSA E. coli
27.45 35.69
24.11 55.12
29.39 26.12
65.17 58.96
0.59 0.04
0.91 1.82
E. coli (MIC: 0.04 mM). Four diterpenoids 8e11 showed moderate antibacterial activities with MIC values of 24.11e65.17 mM against MRSA and 26.12e58.96 mM against E. coli. The positive control gentamicin gave MIC 0.91 mM against MRSA and 1.82 mM against E. coli. 3. Conclusion Marine-sourced actinomycetes of the Streptomyces genus are one of the richest producers of bioactive natural products. In this study, the culture of marine actinomycete Streptomyces sp. ZZ820 in SGYC liquid medium produced 12 compounds, including previously undescribed streptoprenylindoles AeC (1e3), 18-acetyl-cyclooctatin (8), 5,18-dedihydroxy-cyclooctatin (9), and 5-dehydroxycyclooctatin (10). Structures of the new compounds were determined by extensive spectroscopic analyses, ECD calculation, and the Mosher's method. Streptoprenylindoles A and B (1 and 2) are enantiomers that were separated through the preparation of their Mosher esters. The four cyclooctatin diterpenoids (8e11) showed moderated activities in inhibiting the growth MRSA and E. coli with MIC values of 24.11e65.17 mM and the known chromomycin A3 (12) showed potent antibacterial activities against MRSA (MIC: 0.59 mM) and E. coli (MIC: 0.04 mM), which should be the primary contributor to the antibacterial activities observed for the crude extract.
4.2. Isolation and taxonomic identity of strain ZZ820 Strain ZZ820 was isolated from a soil sample collected from sea coastal located at 30 020 3700 N and 121530 2100 E in East China Sea close to Zhoushan Archipelago (Zhejiang, China) on August 16, 2016. Briefly, the dried soils (1.0 g) were diluted with sterile water to make dilutions of 103, 104, and 105 g/mL. Each dilution (200 mL) was covered on the surface of Gauze's solid medium and then incubated at 28 C for 7 days. The single colony (ZZ820) from the 103 g/mL dilution was picked with sterile needles and transferred to a Gauze's-agar plate. After another 7 days of growth at 28 C, the single colony (ZZ820) that grew well was transferred onto Gauze's agar slants and stored at 4 C for further study. The 16S rDNA analysis of strain ZZ820 was performed by Legenomics (Hangzhou, China) and its DNA sequence using BLAST (nucleotide sequence comparison) was compared to the GenBank database. The 16S rDNA sequence of strain ZZ820 has been deposited in GenBank (accession number: MH388488). The voucher strain of Streptomyces sp. ZZ820 (Streptomycetaceae) was preserved at the Laboratory of Institute of Marine Biology, Ocean College, Zhoushan campus, Zhejiang University, Zhoushan, China. 4.3. Large cultivation of strain ZZ820
4. Experimental section
Colonies of strain ZZ820 from the Gauze's agar plate were inoculated into 500 mL Erlenmeyer flasks, each containing 250 mL of sterile GYM liquid medium and then incubated at 28 C for 3 days on a rotary shaker (180 rpm) to produce seed broth. The seed broth (10 mL) was transferred into a 500 mL Erlenmeyer flask containing 250 mL sterile SGYC medium. All flasks were placed on rotary shakers at 180 rpm for incubation at 28 C for 10 days. A total of 40 L culture was prepared for this study.
4.1. General experimental procedures
4.4. Extraction and isolation of compounds 1e12
UV and IR spectra were recorded on a METASH UV-8000 (Shanghai METASH Instruments Co. Ltd., China) and a Nicolet™ IS™ 10 FT-IR spectrometer (Thermo Fisher Scientific), respectively. ECD spectra were obtained on a JASCO J-815 spectropolarimeter. Optical rotation was measured on a RUDOLPH AutopolⅠAutomatic polarimeter. HRESIMS data were acquired on an Agilent 6230 TOF LC/MS spectrometer. NMR spectra were acquired on a Bruker 500 spectrometer or a JEOL 600 spectrometer using standard programs and acquisition parameters and chemical shifts were expressed in d (ppm). Octadecyl-functionalized silica gel (ODS, Cosmosil 75C18Prep, Nacalai Tesque Inc., Japan), silica gel (100e200, Qingdao Haiyang Chemical Co., Qingdao, China), and Diaion HP-20 (Mitsubishi Chemical, Japan) were used for column chromatography. HPLC separation was performed on a CXTH LC-3000 preparative HPLC system (Beijing Chuangxin tongheng Science & Technology Co. Ltd., China) using a CT-30 column (Fuji-C18, 280 30 mm, 10 mm) or an Agilent 1260 HPLC system using Agilent Zorbax SB-C18 columns (250 9.2 mm, 5 mm, or 250 4.6 mm, 5 mm). Mosher reagents were obtained from Aladdin Industrial Corporation (Shanghai, China). All solvents used for this study were purchased from the Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 and Escherichia coli ATCC 25922 were gifts from Drs. Zhongjun Ma and Pinmei Wang, respectively. Gentamicin (99.6%) was purchased from Meilune Biotechnology Co. Ltd. (Dalian, China). GYM medium (glucose 4 g, yeast extract 10 g, malt extract 10 g, tap water 1.0 L) and SGYC medium (soluble starch 20 g, glucose 20 g, yeast extract 10 g, corn flour 3 g, KH2PO4 0.5 g, MgSO4$7H2O 0.5 g, CaCO3 2 g, sea
The 40 L culture of strain ZZ820 was centrifuged to yield supernatant and mycelia. The mycelia were extracted with MeOH three times to give mycelium extract. The supernatant was absorbed onto a HP-20 column eluting with water and then MeOH to obtain crude MeOH supernatant extract. The mycelium and supernatant extracts were combined and then partitioned with EtOAc three times to afford a crude extract (4.2 g). This crude extract (4.2 g) was subjected to a column of silica gel eluting with mixed solvents of cyclohexane/EtOAc (10/1, 5/1, 3/1, 2/1, 1/1, v/v), EtOAc, and MeOH to afford seven fractions (Frs. AeG) based on the results of TLC analysis. By using CXTH LC-3000 preparative HPLC (column: Fuji-C18 CT-30, 280 30 mm, 10 mm; flow rate: 10 mL/min; UV detection: 210 nm), compound 9 (58.3 mg, tR 30 min, MeOH/H2O, 98/2) was purified from Fr. A and compound 6 (3.5 mg, tR 30 min, MeOH/H2O, 77/23) from Fr. B. Compound 4 (2 mg, tR 29 min, ACN/ H2O, 63/37) was obtained from Fr. C by Agilent 1260 HPLC purification (column: Agilent Zorbax SB-C18, 250 9.2 mm, 5 mm; flow rate: 1 mL/min; UV detection: 220 nm). Fr. D was fractionated by an ODS column eluting with 80%, 90%, and 100% MeOH to give three subfractions D1eD3. By using the same SB-C18 column and flow rate with a UV detection of 220 nm for 7 and 210 nm for 8, 10, and 11, each of D1eD3 was purified to afford 11 (24.2 mg, tR 21 min, MeOH/ H2O, 90/10), 8 (15 mg, tR 25 min, MeOH/H2O, 90/10), and 10 (21.8 mg, tR 27 min, MeOH/H2O, 90/10), respectively, and compound 7 (4 mg, tR 28 min, MeOH/H2O, 60/40) was obtained from Fr. E. In the same way, Fr. F was separated by an ODS column eluting with 40%, 50%, and 100% MeOH to give three subfractions F1eF3. Further purification of F2 and F3 by the same SB-C18 column and
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flow rate with UV detection of 220 nm gave 3 (4.8 mg, tR 39 min, MeOH/H2O, 47/53) and 5 (1.5 mg, tR 43 min, MeOH/H2O, 47/53) from F2 and a mixture of 1 and 2 (14.2 mg, tR 45 min, MeOH/H2O, 47/53) from F3. Fr. G was also separated by ODS column eluting with 65%, 75%, and 100% MeOH to give G1eG3. Compound 12 (21 mg, tR 29 min, ACN/0.1% HOAc in water, 49/51) was purified from G1 by HPLC using the SB-C18 column (flow rate: 1 mL/min; UV detection: 278 nm). In order to separate compounds 1 and 2, the mixture (14.2 mg) of 1 and 2 was dissolved in 1 mL pyridine and then treated with 90 mL S-MTPA-Cl for 2 h at room temperature. The reaction was terminated by an addition of 0.5 mL MeOH. The resulting mixture was dried in vacuo to give a residue, which was purified by HPLC (column: SB-C18 250 4.6 mm, 5 mm; mobile phase: MeOH/H2O, 60/40; flow rate: 0.8 mL/min; UV detection: 220 nm) to give their R-MTPA esters (1r, tR 34.4 min; 2r, tR 37.2 min). Each of the R-MTPA ester (1r or 2r) was in turn hydrolyzed by LiOH to furnish pure compound 1 or 2. Briefly, compound 1r or 2r (each 6 mg) was dissolved in a mixture of MeOH/H2O (3:1, 2 mL) and then treated with 1 mL 3 N LiOH in MeOH/H2O (3:1) under stirring at room temperature for 12 h. The reaction mixture was neutralized with 3 N HCl and then concentrated under reduced pressure to give a residue, which was purified by HPLC (column: SB-C18 250 4.6 mm, 5 mm; mobile phase: MeOH/H2O, 45/55; flow rate: 0.8 mL/min; UV detection: 220 nm) to give 1 (5.0 mg, tR 11.6 min) or 2 (4.5 mg, tR 11.6 min). 4.4.1. Streptoprenylindole A (1) White amorphous powder; molecular formula C15H18N2O2; [a]20 D þ47.4 (c 0.10, MeOH); UV (MeOH) lmax (log ε) 203 (3.95), 222 (4.17), 274 (3.36), 280 (3.36) nm; IR (MeOH) nmax 3404, 2975, 2931, 2252, 1639, 1454, 1348, 1064 cm1; 13C NMR data (125 MHz, in MeOH-d4), see Table 1, 1H NMR data (500 MHz, in MeOH-d4), see Table 2; HRESIMS m/z [MþH]þ 259.1446 (calcd for C15H19N2O2, 259.1447), [MþNa]þ 281.1262 (calcd for C15H18N2NaO2, 281.1266), and [M H]e 257.1296 (calcd for C15H17N2O2, 257.1290). 4.4.2. Streptoprenylindole B (2) White amorphous powder; molecular formula C15H18N2O2; [a]20 D 40.0 (c 0.10, MeOH); UV (MeOH) lmax (log ε) 203 (3.95), 222 (4.17), 274 (3.36), 280 (3.36) nm; IR (MeOH) nmax 3397, 2977, 2927, 2252, 1627, 1456, 1343, 1066 cm1; 13C NMR data (125 MHz, in MeOH-d4), see Table 1, 1H NMR data (500 MHz, in MeOH-d4), see Table 2; HRESIMS m/z [MþNa]þ 281.1261 (calcd for C15H18N2NaO2, 281.1266) and [M H]e 257.1294 (calcd for C15H17N2O2, 257.1290). 4.4.3. Streptoprenylindole C (3) White amorphous powder; molecular formula C15H21NO3; [a]20 D 18.8 (c 0.10, MeOH); UV (MeOH) lmax (log ε) 205 (4.01), 226 (4.34), 283 (3.54) nm; IR (MeOH) nmax 3306, 2922, 2852, 1454, 1339, 1064, 1029 cm1; 13C NMR data (125 MHz, in MeOH-d4), see Table 1, 1 H NMR data (500 MHz, in MeOH-d4), see Table 2; HRESIMS m/z [M H]e 262.1446 (calcd for C15H20NO3, 262.1443). 4.4.4. 18-Acetyl-cyclooctatin (8) Colorless oil; molecular formula C22H36O4; [a]20 D þ108.6 (c 0.10, MeOH); ECD (9.5 mg/L, MeOH) lmax (Dε) 204 (þ38.5) nm; UV (MeOH) lmax (log ε) 203 (3.93) nm; IR (MeOH) nmax 3315, 2359, 2333, 1738, 1464, 1367, 1249, 1109 cm1; 13C NMR data (150 MHz, in DMSO‑d6), see Table 1, 1H NMR data (600 MHz, in DMSO‑d6), see Table 3; HRESIMS m/z [MþNa]þ 387.2508 (calcd for C22H36NaO4, 387.2511) and [2 MþNa]þ 751.5109 (calcd for C44H72NaO8, 751.5125).
4.4.5. 5,18-Dedihydroxy-cyclooctatin (9) Colorless oil; molecular formula C20H34O; [a]20 D þ105.5 (c 0.10, MeOH); ECD (13.0 mg/L, MeOH) lmax (Dε) 204 (þ27.8) nm; UV (MeOH) lmax (log ε) 203 (3.79) nm; IR (MeOH) nmax 3438, 2361, 2341, 1460, 1375, 1293, 926 cm1; 13C NMR data (150 MHz, in DMSO‑d6), see Table 1, 1H NMR data (600 MHz, in DMSO‑d6), see Table 3; HRESIMS m/z [MþНeН2Ο]þ 273.2576 (calcd for C20H33, 273.2582). 4.4.6. 5-Dehydroxy-cyclooctatin (10) Colorless oil; molecular formula C20H34O2; [a]20 D þ157.5 (c 0.10, MeOH); ECD (10.0 mg/L, MeOH) lmax (Dε) 205 (þ31.5) nm; UV (MeOH) lmax (log ε) 204 (3.94) nm; IR (MeOH) nmax 3434, 2364, 2339, 1458, 1379, 1299, 1109 cm1; 13C NMR data (150 MHz, in DMSO‑d6), see Table 1, 1H NMR data (600 MHz, in DMSO‑d6), see Table 3; HRESIMS m/z [MþNa]þ 329.2457 (calcd for C20H34NaO2, 329.2457). 4.5. MTPA esterification of 1, 2, and 8 A solution of compound 1 (2.0 mg) in 2 mL anhydrous pyridine was divided into two equal portions. R-MTPA-Cl or S-MTPA-Cl (each 45 mL) was added to each portion. Each mixture was stirred for 2 h at room temperature and the reaction was terminated by an addition of 0.5 mL MeOH. Each reaction mixture was concentrated in vacuo to give a residue, which was purified by HPLC (column: SBC18 250 4.6 mm, 5 mm; mobile phase: MeOH/H2O, 60/40; flow rate: 0.8 mL/min; UV detection: 220 nm) to give 1s (S-MTPA ester of 1, 0.5 mg, tR 37.2 min) or 1r {R-MTPA ester of 1, 0.7 mg, tR 34.4 min, HRESIMS m/z [MþNa]þ 497.1661 (calcd for C25H25F3N2NaO4, 497.1664)} for NMR analysis. Compound 2 (2.0 mg) was also treated by R-MTPA-Cl or S-MTPA-Cl to afford 2s (S-MTPA ester of 2, 0.7 mg, tR 34.4 min) or 2r {R-MTPA ester of 2, 0.5 mg, tR 37.2 min, HRESIMS m/z [MþNa]þ 497.1660 (calcd for C25H25F3N2NaO4, 497.1664)} for NMR analysis. The 1H NMR data for 1s, 1r, 2s, and 2r are summarized in Table S5. In the same way, compound 8 (2.0 mg) was reacted with R-MTPA-Cl or S-MTPA-Cl to afford 8s (S-MTPA ester of 8, 1.8 mg, tR 24.7 min, MeOH/H2O, 95/5; flow rate, 1.0 mL/min) or 8r (R-MTPA ester of 8, 1.9 mg, tR 23.6 min) for NMR analysis. The 13C and 1H NMR data for 8s and 8r are summarized in Table S6. 4.6. ECD calculation Monte Carlo conformational searches were carried out by means of the Spartan's 10 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmann-population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31 þ g (d, p) level in MeOH using the CPCM polarizable conductor calculation model (Figs. S131 and S132 and Tables S7 and S9). The theoretical calculation of ECD was conducted in MeOH using Time-dependent Density functional theory (TD-DFT) at the B3LYP/6-311 þ g (d, p) level for all conformers of compounds 8 and 9 (Tables S8 and S10). Rotatory strengths for a total of 50 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma ¼ 0.3 eV. 4.7. Antibacterial assay The antibacterial activities of compounds 1e12 against the growth of MRSA and E. coli were determined by the micro broth dilution method as described in previous publication [27]. Gentamicin was used as a positive control and DMSO was used as a
W. Yi et al. / Tetrahedron 75 (2019) 1186e1193
negative control. Conflicts of interest The authors declare no conflict of interest. Acknowledgments This study was supported by the National Key R&D Program of China (No. 2017YFE0102200) and the National Natural Science Foundation of China (81773587, 81773769, and 81273428). We appreciate Dr. Jianyang Pan (Pharmaceutical Informatics Institute of Zhejiang University) for performing the NMR spectrometry. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.tet.2019.01.025. References [1] M. Kamjam, P. Sivalingam, Z. Deng, K. Hong, Deep sea actinomycetes and their secondary metabolites, Front. Microbiol. 8 (2017) 760. [2] C. Zhao, T. Zhu, W. Zhu, Chin. J. Org. Chem. 33 (2013) 1195e1234. [3] U.R. Abdelmohsen, K. Bayer, U. Hentschel, Nat. Prod. Rep. 31 (2014) 381e399. [4] P. Chen, L. Zhang, X. Guo, X. Dai, L. Liu, L. Xi, J. Wang, L. Song, Y. Wang, Y. Zhu, L. Huang, Y. Huang, Front. Microbiol. 7 (2016) 1340. [5] A.S. Azman, I. Othman, S.S. Velu, K.G. Chan, L.H. Lee, Front. Microbiol. 6 (2015) 856. [6] K. Hong, A.H. Gao, Q.Y. Xie, H. Gao, L. Zhuang, H.P. Lin, H.P. Yu, J. Li, X.S. Yao, M. Goodfellow, J.S. Ruan, Mar. Drugs 7 (2009) 24e44. [7] P.G. Williams, Trends Biotechnol. 27 (2009) 45e52. [8] P. Manivasagan, J. Venkatesan, K. Sivakumar, S.K. Kim, Microbiol. Res. 169
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