Identification and Proposed Relative and Absolute Configurations of

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Identification and Proposed Relative and Absolute Configurations of Niphimycins C−E from the Marine-Derived Streptomyces sp. IMB7-145 by Genomic Analysis Yuanyuan Hu,†,§ Mian Wang,†,§ Chunyan Wu,†,‡ Yi Tan,† Jiao Li,† Xiaomeng Hao,† Yanbo Duan,† Yan Guan,† Xiaoya Shang,‡ Yiguang Wang,† Chunling Xiao,† and Maoluo Gan*,† †

Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University, Beijing 100191, People’s Republic of China S Supporting Information *

ABSTRACT: Analysis of the whole genome sequence of Streptomyces sp. IMB7-145 revealed the presence of seven type I polyketide synthase biosynthetic gene clusters, one of which was highly homologous to the biosynthetic gene cluster of azalomycin F. Detailed bioinformatic analysis of the modular organization of the PKS gene suggested that this gene is responsible for niphimycin biosynthesis. Guided by genomic analysis, a large-scale cultivation ultimately led to the discovery and characterization of four new niphimycin congeners, namely, niphimycins C−E (1−3) and 17-O-methylniphimycin (4). The configurations of most stereocenters of niphimycins have not been determined to date. In the present study, the relative configurations were elucidated by spectroscopic analysis, including J-based analysis and the CNMR database method. Further, the full absolute configurations of niphimycins were completely proposed for the first time based on biosynthetic gene cluster analysis of the ketoreductase and enoylreductase domains for hydroxy- and methyl-bearing stereocenters. Compounds 1, 3, 4, and niphimycin Iα (5) showed antimicrobial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci (MIC: 8−64 μg/mL), as well as cytotoxicity against the human HeLa cancer cell line (IC50: 3.0−9.0 μM). In addition, compounds 1 and 5 displayed significant activity against several Mycobacterium tuberculosis clinical isolates (MIC: 4−32 μg/mL).

M

actinomycetes,11−13 the extract of Streptomyces sp. IMB7-145 was found to display significant antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) pathogens. Previously, we identified six elaiophylins from the culture extracts of this strain using a PCR screening-guided method.13 In order to further mine the secondary metabolite potential and identify new metabolites against drug-resistant pathogens, we sequenced the genome of Streptomyces sp. IMB7-145. A search of the genome sequence using antiSMASH 4.014 identified seven type I polyketide synthase (PKS) gene clusters (Table S1, Supporting Information), including the known gene clusters of nigericin15 and elaiophylin.16 One of the unknown PKS gene clusters showed high similarity to the biosynthetic gene cluster (BGC) of azalomycin F in Streptomyces violaceusniger DSM 413717,18 and Streptomyces sp. 211726.19 Detailed bioinformatic analysis of the modular organization in this gene cluster suggested that

icrobial natural products represent essential sources of chemical diversity for drug discovery, particularly that of antibiotics.1 The continued emergence of bacterial resistance over the last several decades has marginalized many important antibiotics, necessitating the urgent development of new antibiotics to combat resistant pathogens.2 Marine actinomycetes have been proven to be prolific sources of bioactive and structurally intriguing natural products over the last two decades.3,4 At the same time, advances in genome sequencing, along with an ever-increasing knowledge of biosynthetic machinery, have spawned a new field of genomics-guided discovery of microbial natural products.5,6 Genomics-guided drug discovery utilizes the biosynthetic genetic information found in the genome of microbes to predict metabolite molecular frameworks; this may include the precise chemical structure and configurations.5,7,8 This genome-directed strategy has been successfully applied to natural product discovery, leading to the discovery of novel chemical entities of potential clinical utility.8−10 During our ongoing program to discover new antibiotics against drug-resistant pathogen bacteria from marine-derived © XXXX American Chemical Society and American Society of Pharmacognosy

Received: October 13, 2017

A

DOI: 10.1021/acs.jnatprod.7b00859 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

The first PKS gene (npmA) is nearly identical (92% protein identity) to the corresponding gene (azlA) in the azalomycin cluster, both encoding a lone acyl carrier protein (ACP) domain in the loading domain and module 1 of the polyketide chain extension.17−19,26 As only 20 extension modules in the cluster mediate the required 21 cycles of polyketide elongation, we predicted that module 1 in NpmA, like its AzlA counterpart, iteratively catalyzes the first and second cycles of chain extension.19,26 A comparison of the arrangement of the npm and azl gene clusters suggests that npmB and npmC are equivalents of the azlB gene. However, npmB and npmC contain one extra module (module 3) consisting of ketosynthase (KS), acyltransferase (AT), dehydratase (DH), enoylreductase (ER), ketoreductase (KR), and ACP domains. The presence of the extra module in the npm gene is consistent with the presence of the additional saturated propionate unit (C-35, C-36, and Me-36) in the side chain of niphimycin. As in the case of azlB, module 2 of npmB lacks an ER domain required for the full reduction of an enoyl thioester at this stage (Figure 2). A plausible explanation is that the missing function is provided by an ER domain in the neighboring module.26 The arrangement of the remaining PKS genes in the npm cluster displays high homology to the corresponding PKS genes in the azl cluster, with the exception that the last gene (npmI) encoding the terminal module contains an unusual reductive loop comprising one DH and two KR domains. A type I PKS gene encoding this type of terminal module with two KR domains has been reported in the genome of S. melanosporofaciens and S. violaceusniger Tu 4113, but for uncharacterized secondary metabolites (SED16733 and WP_014057316, 99% and 85% identity, respectively). Although predicted to be active (Figures S2 and S3), the DH domain and the second KR domain in the terminal module appear to be skipped, as is the ER domain in the first extension cycle in the Npm and azl clusters.19,26 This leads to the generation of a C-3 hydroxy in the niphimycin structure. Such skipping of an apparently active domain in a cis-AT PKS is well precedented.27,28 The npm cluster additionally contains a set of genes involved in the biosynthesis of 4-guanidinobutyryl-CoA, a common starter unit for guanidine-containing macrocyclic polyketides such as azalomycin F, kanchanamycin, and clethramycin.17,18 The incorporation mechanism of 4-guanidinobutyryl-CoA into the polyketide backbone has been characterized for the azalomycin F- and clethramycin-producing strain S. violaceusniger DSM 4137. The 4-guanidinobutyryl precursor originates from arginine via a series of reactions including decarboxylation and hydrolysis catalyzed by an L-arginine monooxygenase (AM), a 4-guanidinobutyramide hydrolase (AH), a 4-guanidinobutanoate-CoA ligase (CoL), and a 4-guanidinobutyryl-CoA ACP acyltransferase (AT).17 Npm6 (CoL), Npm7 (AT), and Npm20 (AH), which were proposed to catalyze these reactions, share high amino acid sequence identity with the homologous gene

it was responsible for the biosynthesis of the azalomycin-related macrolide niphimycin.20−22 Guided by genome analysis, we identified four new niphimycin analogues, namely, niphimycins C−E (1−3) and 17-O-methylniphimycin (4), together with the known niphimycin Iα (5) and 19-O-malonylniphimycin (6),23 from the cultures of strain IMB7-145. Although the planar structures of the niphimycins were established more than 30 years ago, their absolute configurations have not been completely resolved to date.20−22,24 On the basis of a combination of detailed spectroscopic and bioinformatics analyses, we propose the full relative and absolute configurations of the niphimycins. Herein, we report the identification of the niphimycin biosynthetic gene cluster, elucidation of the structures of the new niphimycins, and characterization of their bioactivities.

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RESULTS AND DISCUSSION Identification and Characterization of the Niphimycin Biosynthetic Gene Cluster. The complete genome sequence of S. sp. IMB7-145 was obtained via de novo sequencing using single molecule real-time technology (SMRT, Pacbio RSII)25 combined with the Illumina HiSeq 4000 sequencing system. Gene clusters with putative roles in secondary metabolism were identified using antiSMASH 4.0,14 which revealed a PKS gene cluster highly homologous to the azl gene cluster responsible for the biosynthesis of azalomycin F in S. violaceusniger DSM 413717,18 and S. sp. 211726 (Figures 1 and S1).19 This cluster (named npm) spans a 132.4 kbp DNA region and contains 31 individual open reading frames (ORFs), in which nine large genes (npmA−npmI) encode a 20-module type I PKS. The putative functions of each gene were assigned by comparing the deduced amino acid sequence with the Azl proteins and other bacterial homologues (Table S2). Detailed bioinformatics analysis revealed that the predicted domain and module architecture were in good agreement with the assembly of the polyketide skeleton of niphimycin (Figure 2).

Figure 1. Graphical comparison of the niphimycin biosynthetic gene cluster (npm) and the azalomycin biosynthetic gene cluster (azl). B



Article

Figure 2. Proposed model for niphimycin biosynthesis. Shown is modular organization of npm PKS with catalytic domains. The configurations of the stereocenters were predicted based on sequence analysis of KR and ER domains. The black circles mark domains presumed to be nonfunctional. The dashed circles represent the missing domains that are required for the assembly of the niphimycin skeleton. ATa, acyltransferase incorporating an acetate unit; ATp, acyltransferase incorporating a propionate unit; ACP, acyl carrier protein; DH, dehydratase; ER, enoylreductase; KS, ketosynthase; KR, ketoreductase; KR*: B1 type (2R,3R); KR#: A1 type (2R, 3S); TE, thioesterase.

a tBLASTn search of the S. sp. IMB7-145 genome sequence revealed a gene with high identity to an arginine monooxygenase gene located in the putative clethramycin BGC

products in the azl cluster (Azl4, 94%; Azl5, 87%; and Azl13, 93%, respectively, Table S2).19 Although the arginine monooxygenase gene has not been identified in the npm cluster, C



Article

Table 1. 1H NMR Spectroscopic Data for Compounds 1−4 in CD3OD

a

1

2

3

4

1

2

3

4

δH, mult. [J in (Hz)]








2.45, dq (9.4, 6.6) 4.10, dd (8.4, 9.0) 5.42, dd (15.6, 9.0) 5.69, dd (15.6, 7.8) 2.33, m

7 8

2.44, dq (8.5, 7.0) 4.09, dd (8.0, 8.5) 5.42, dd (15.5, 8.0) 5.69, dd (15.5, 8.0) 2.31, dqd (8.0, 7.0, 3.0) 3.77, m 1.70, m; 1.51, m

9

3.76, m

10 11 12

1.51, m 3.87, m 1.60, m; 1.37, m

13 14 15 16

1.44, 1.60, 3.87, 1.90,

18 19

3.32, d (9.5) 3.86, m

20

1.89, m; 1.32, m

21

4.16, tt (9.0, 2.5)

22

1.70, m; 1.56, m

23 24

3.86, m 1.83, m; 1.70, m

25 26

5.23, m 1.72, m; 1.53, m

27

3.95, m

3.70, d (9.0) 5.19, td (10.2, 4.2) 2.05, m; 1.42, m 4.22, tt (12.0, 3.0) 1.56, m; 1.33, m 3.89, m 1.89, m; 1.70, m 5.30, m 1.73, m; 1.55, m 3.94, m

28 29

1.53, m 4.04, dd (7.0, 7.0) 5.64, dd (15.5, 7.0)

1.52, m 4.03, dd (7.8, 7.2) 5.64, dd (15.0, 7.2)

no. 2 3 4 5 6

30

m; 1.32, m m m m; 1.78, m

3.79, m 1.70, m; 1.51, m 3.75, td (7.8, 3.0) 1.50, m 3.88, m 1.60, m; 1.35, m 1.30, m 1.60, m 3.88, m 1.90, m; 1.81, m

2.42, dq (8.4, 7.8) 4.07, m

2.44, dq (8.4, 7.2) 4.09, t (8.4)

5.40, dd (15.6, 7.8) 5.73, dd (15.6, 7.8) 2.28, m

5.42, dd (15.6, 8.4) 5.70, dd (15.6, 8.4) 2.31, m

3.71, m 1.76, m; 1.45, m

3.74, m 1.73, m; 1.49, m

3.73, m

3.75, m

1.50, m 3.92, m 1.63, m; 1.35, m

1.50, m 3.90, m 1.63, m; 1.32, m

1.49, m; 1.37, m 1.78, m 5.12, m 2.21, dd (15.0,7.8); 1.78, m 3.18, d (9.6) 3.83, m

1.40, 1.62, 3.79, 1.90,

3.50, d (9.0) 3.88, m

1.89, m; 1.27, m

1.91, m; 1.30, m

4.06, m

3.79, m

1.77, m; 1.69, m

1.80, m; 1.69, m

5.28, m 1.85, m; 1.72, m

5.26, tt (8.4, 4.2) 1.80, m; 1.72, m

3.84, m 1.60, m; 1.38, m

3.88, m 1.60, m; 1.38, m

4.16 brd (10.2)

4.14, dt (10.8, 3.0) 1.52, m 4.06, dd (7.8, 6.6) 5.66, dd (15.0, 6.6)

1.50, m 4.05, m 5.66, dd (15.6, 6.6)

no. 31

6.17, dd (15.5, 10.0)

32

6.06, dd (15.0, 10.0)

33

36 37

5.54, dd (15.0, 9.0) 2.55, ddq (9.0, 8.0, 7.0) 4.75, dd (8.0, 4.0) 1.87, m 1.36, m; 0.93, m

38 39

1.57, m 1.30, m; 1.06, m

40 41

1.34, m 1.98, quint (7.0)

42

5.48, dt (15.5, 7.0) 5.43, dt (15.5, 7.0) 2.07, q (7.0) 1.65, quint (7.0)

34 35

m; 1.34, m m m m; 1.72, m

43 44 45 46 47 48 49 50 51 52 53 54 56 2′ 2″ OMe

3.16, t (7.0) 1.02, d (7.0) 1.08, d (6.5) 0.88, d (7.0) 0.91, d (7.0) 0.84, d (7.0) 1.02, d (7.0) 0.90, d (6.5) 0.87, d (7.0) 2.84, s 3.24, d (14.8); 3.21, d (14.8)

6.17, dd (15.0, 10.2) 6.06, dd (15.0, 10.2) 5.53, dd (15.0, 9.0) 2.55, m 4.75, dd (9.0, 4.2) 1.89, m 1.35, m; 0.93, m 1.55, m 1.30, m; 1.04, m 1.34, m 1.98, quint (7.0) 5.48, dt (15.0, 7.2) 5.43, dt (15.0, 7.2) 2.08, q (7.2) 1.65, quint (7.2) 3.17, t (7.2) 1.01, d (6.6) 1.09, d (6.6) 0.88, d (6.0) 0.92, d (6.6) 0.83, d (6.6) 1.02, d (7.2) 0.91, d (6.0) 0.86, d (6.6) 2.85, s 3.22, m 3.22, m

6.19, dd (15.6, 10.2)

6.18, dd (15.0, 10.8)

6.06, dd (15.0, 10.2)

6.06, dd (15.0, 10.2)

5.49, dd (15.0, 9.0) 2.53, ddq (9.0, 8.4, 6.6) 4.75, dd (8.4, 3.0) 1.92, m 1.34, m; 0.91, m

5.51, dd (15.0, 9.0) 2.54, m 4.75, dd (7.8, 3.0) 1.89, m 1.35, m; 0.91, m

1.57, m 1.31, m; 1.06, m

1.57, m 1.29, m; 1.03, m

1.36, m 1.98, quint (6.6)

1.35, m 1.98, m

5.48, dt (15.0, 6.6) 5.42, dt (15.0, 6.0) 2.07, q (7.2) 1.65, m

5.49, dt (15.0, 6.6) 5.45, dd (15.0, 6.6) 2.08, q (7.2) 1.65, m

3.16, t (7.2) 1.00, d (7.2) 1.06, d (7.2) 0.85, d (6.6) 0.91, d (7.2) 0.86, d (6.6) 1.02, d (6.6) 0.92, d (6.6) 0.87, d (6.0) 2.84, s 3.24, d (12.0); 3.22, d (12.0) 3.24, d (12.0); 3.22, d (12.0)

3.16, t (7.2) 1.01, d (7.2) 1.08, d (6.6) 0.88, d (6.6) 0.93, d (6.6) 0.84, d (7.2) 1.02, d (7.2) 0.92, d (6.6) 0.87, d (6.6) 2.83, s 3.24, d (12.0); 3.22, d (12.0)

3.19, s

a1 H NMR data were recorded at 500 MHz for 1 and 600 MHz for 2−4. The assignments were based on 2D NMR (COSY, HSQC, and HMBC) experiments.

the malonyl acylation reaction. As discussed below and as was also observed by other researchers,29 more malonyl goups in the molecule resulted in decreased antibacterial activities. Presumably, this gene is related to self-resistance and widely present in actinomycete bacteria. Isolation and Structure Elucidation of Niphimycins. To obtain the expression product of this gene cluster, strain IMB7-145 was cultured in 14 different media, with or without supplementation of 3% artificial sea salts. After harvesting, the culture broth was adsorbed on XAD7HP resin and then washed with distilled H2O before acetone elution. The acetone extracts were subjected to HPLC-ESIMS analysis, which showed that extracts from several media produced compounds with a UV absorption maximum at 232 nm and MS peaks at m/z 1142 [M + H]+ and 1228 [M + H]+ (Figures S7 and S8).

(Figure S6). The 4-guanidinobutyryl-CoA biosynthetic genes were shown to be distributed across two different loci in the strain IMB7-145. A similar distribution has also been reported in the azalomycin F-producing strains S. violaceusniger DSM 413717 and S. sp. 211726.19 As is the case in the azl cluster, the malonyl transferase gene responsible for the malonyl acylation of the hydroxy groups at C-15/19/23/25 is not present in the npm gene cluster of strain IMB7-145. This gene may be elsewhere in the genome, but was not identified. Hong and co-workers found that the heterologous expression of the azl gene cluster in S. lividans led to production of the same azalomycins F3a, F4a, and F5a with the malonyl side chain at C-23 as those from the producing strain S. violaceusniger DSM4137.26 It was deduced that the heterologous host could provide the appropriate enzyme to catalyze D



Article

Table 2. 13C NMR Spectroscopic Data for Compounds 1−4 in CD3ODa 1

2

3

4

1

2

3

4

no.

δC, type

δC, type

δC, type

δC, type

no.

δC, type

δC, type

δC, type

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

176.9, C 47.9, CH 76.1, CH 132.6, CH 136.4, CH 43.4, CH 75.8, CH 39.5, CH2 75.0, CH 44.7, CH 72.4, CH 33.3, CH2 30.1, CH2 40.5, CH 72.1, CH 42.0, CH2 99.9, C 77.7, CH 69.7, CH 41.3, CH2 66.3, CH 44.4, CH2 65.9, CH 43.7, CH2 71.2, CH 40.4, CH2 68.9, CH 45.4, CH 75.2, CH 134.9, CH 132.1, CH 131.9, CH




33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 1′ 2′ 3′ 1″ 2″ 3″ OMe

136.9, CH 40.6, CH 80.1, CH 32.6, CH 42.4, CH2 30.7, CH 37.3, CH2 27.6, CH2 33.9, CH2 132.9, CH 129.9, CH 30.7, CH2 29.9, CH2 42.0, CH2 15.2, CH3 17.3, CH3 10.7, CH3 15.4, CH3 11.1, CH3 18.1, CH3 14.9, CH3 20.6, CH3 158.2, C 28.4, CH3 171.4, C 46.2, CH2 174.1, C

136.9, CH 40.6, CH 80.2, CH 32.7, CH 42.4, CH2 30.8, CH 37.3, CH2 27.7, CH2 33.9, CH2 132.9, CH 129.9, CH 30.7, CH2 29.9, CH2 42.0, CH2 15.2, CH3 17.2, CH3 10.7, CH3 15.5, CH3 11.2, CH3 18.1, CH3 14.9, CH3 20.6, CH3 158.3, C 28.4, CH3 170.8, C NDb 173.6, C 171.2, C NDb 173.8, C

136.9, CH 40.7, CH 79.5, CH 32.5, CH 42.5, CH2 30.6, CH 37.6, CH2 27.8, CH2 33.9, CH2 132.9, CH 129.9, CH 30.7, CH2 29.9, CH2 42.0, CH2 15.0, CH3 16.8, CH3 10.5, CH3 14.3, CH3 11.1, CH3 17.8, CH3 14.7, CH3 20.4, CH3 158.2, C 28.4, CH3 170.6, C NDb 173.8, C 171.4, C NDb 173.9, C

136.8, CH 40.6, CH 79.8, CH 32.6, CH 42.5, CH2 30.6, CH 37.5, CH2 27.7, CH2 33.9, CH2 133.0, CH 129.9, CH 30.7, CH2 29.9, CH2 42.0, CH2 14.9, CH3 16.8, CH3 10.6, CH3 14.9, CH3 11.2, CH3 17.9, CH3 14.9, CH3 20.5, CH3 158.3, C 28.3, CH3 171.4, C NDb 173.9, C

48.1, CH3

a13

C NMR data were recorded at 125 MHz for 1 and 150 MHz for 2−4. The assignments were based on 2D NMR (COSY, HSQC, and HMBC) experiments. bND: not detected. The signals were not detected due to the facile exchange of H2-2′(2″) with deuterium under the measurement conditions.

These characteristics were consistent with those of niphimycins.20 The highest level of production was found to occur in the M7 medium. Accordingly, large-scale fermentation of strain IMB7-145 was carried out in this medium. Guided by the UV and MS characteristics, further isolation of the fermentation broth by chromatography on XAD7HP resin, silica gel, and Sephadex LH-20, followed by preparative HPLC, yielded six pure compounds. In addition to the known niphimycin Iα (5) and 19-O-malonylniphimycin (6),23 four new niphimycin congeners (1−4) were obtained. Compound 1 was isolated as a white powder; its molecular formula of C59H103N3O18 was established by HRESIMS and NMR data (Tables 1 and 2). Interpretation of the 1D and 2D NMR spectra (COSY and HSQC) revealed the presence of one conjugated 1,3-diene system, two nonconjugated olefinic double bonds, 13 oxygenated methines, eight aliphatic methines, 16 methylenes, eight aliphatic methyl groups, one amino methyl group, and five nonprotonated carbon signals (including one hemiketal carbon at δC 99.9, one guanidinyl carbon at δC 158.2, and three carboxyl or ester carbonyl carbons at δC 171.4− 176.9). Detailed analyses of the COSY and TOCSY spectra enabled the construction of two large spin systems comprising C-2−C-16 and C-18−C-46, as illustrated by bold lines

Figure 3. COSY (bold lines) and key HMBC correlations of niphimycin C (1).

(Figure 3). The HMBC correlations of H-16a/H-16b with C-17/C-18, H-2 with C-1, and H2-46/H3-56 with the guanidinyl carbon C-55 enabled connection of the two large segments through C-17 and extension of the linear polyketide chain from C-1 to C-46, with a methylguanidinyl group attached at C-46. Additionally, the long-range correlation of H-35 with C-1 led to the assembly of a 36-membered macrolide ring. Although the correlation of H-21 with C-17 was not observed in the HMBC spectrum, the remaining one degree of unsaturation required for the molecular formula and the characteristic hemiketal carbon resonance of C-17 (δC 99.9)23 placed an ether linkage E



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been determined.20,24,31 In this report, we resolved the relative configurations of C-2/C-3, C-6/C-7, and C-28/C-29 as anti, anti, and anti, respectively, using J-based configurational analysis (Figure S11).32 In addition, the relative configurations of C-7/C-9/C-10/C-11 were elucidated as syn/anti/syn by application of Kishi’s universal NMR database (Figures S12 and S13).33,34 Similarly, the relative configuration of C-14/C-15 was deduced as anti via comparison of the 13C chemical shift of the methyl (C-50) with that of 4-methylnonan-5-ol isomers (Figure S14).35 The relative configuration of the pyran ring (C-17−C-21) was determined by 1H−1H coupling constants and ROESY correlations, as shown in Figure S15. The large coupling constant (9.5 Hz) between H-18 and H-19 established their trans relationship and, therefore, axial orientation. The ROE correlations of H-18/H-16a and H-18/H-16b indicated that these protons were oriented on the same side of the pyran ring, whereas the ROE correlation of H-19/H-21 demonstrated that they were on the other side of the ring. All of the double bonds were deduced to possess the E-configuration based on their large 3J coupling constants (∼15.0 Hz). In order to determine the absolute configuration of niphimycin, compound 5 was subjected to chemical degradation (NaBH4 reduction and methanolysis), yielding the hydrolyzed products 7 and 8 (Scheme 1). However, our attempts to achieve further periodate oxidation and derivatization (selective hydroxy protection, acetonide derivatization, and Mosher’s ester analysis) were unsuccessful as a result of the complexity of the degradation products, challenges associated with their purification, and scarcity of the natural product. Therefore, we focused on the identified npm gene cluster. During polyketide biosynthesis, the KR domain stereospecifically reduces the β-keto group to generate a hydroxy group; the DH domain subsequently eliminates H2O to form either cis or trans double bonds, depending on the configuration of the preceding hydroxy-bearing stereocenter, and the ER domain stereospecifically reduces the double bond to create an isolated methylbearing center.36 The absolute configurations of the carbons with the α-branching substituents and β-hydroxy groups of polyketides may be accurately predicted according to the crucial conserved amino acid residues in the core KR region.37−39 This technique contributed to the configurational assignment of a number of natural polyketides, many of which have been verified by other chemical methods.8,18,40−44 In the npm gene cluster, KR domains in modules 4, 7, 9, 11, 14, 17, 18, and 20a

between C-17 and C-21 to form a six-membered hemiketal ring. Furthermore, the resonance signals at δC 171.4 (C), 46.2 (CH2), and 174.1 (C) observed in the 13C NMR and DEPT spectra indicated the presence of one malonyl residue in 1. Finally, the HMBC correlation of H-25 (δH 5.23) with the ester carbon C-1′ (δC 171.4) suggested that the malonyl group is located at C-25. Thus, the planar structure of 1 was established as 23-O-demalonyl-25-O-malonylniphimycin and named niphimycin C. Compounds 2 and 3 were assigned the same molecular formula (C62H105N3O21) by their similar HRESIMS and NMR data. This formula revealed the addition of C3H2O3 to the molecular formula of compound 1, suggesting that an extra malonyl group is attached to the polyketide skeleton of 2 and 3. The NMR data of 2 and 3 both showed high similarity to those of 1, except for the presence of the extra signals for a malonyl residue. Comprehensive analyses of 2D NMR data of 2 and 3 (COSY, ROESY, HSQC, and HMBC) revealed that the two malonyl groups were attached at C-19/C-25 in 2 and at C-15/ C-23 in 3 (Figure S9). Therefore, the structures of 2 and 3 were identified as 23-O-demalonyl-19,25-O-dimalonylniphimycin and 15-O-malonylniphimycin, respectively, and the compounds were named niphimycins D (2) and E (3). Compound 4 has the molecular formula C60H105N3O18, as determined by HRESIMS and NMR data. The NMR data of 4 were similar to those of 1. The major difference between the NMR spectra of 4 and 1 was the presence of a new methoxy singlet (δH 3.19 and δC 48.1). The HMBC spectrum showed a heteronuclear correlation between the methoxy singlet (δH 3.19) and C-17 (δC 102.8), thus suggesting that the hemiketal hydroxy at C-17 was replaced by a methoxy group in 4. Furthermore, detailed 2D NMR data analysis revealed that the malonyl residue was substituted at C-23 in 4 (Figure S9). Thus, the structure of 4 was elucidated as 17-O-methylniphimycin. During the isolation process, we have observed that compound 5 could transform to 4 in the MeOH solution in the presence of a trace of acetic acid, suggesting that 4 was an artifact arising from ketalization of 5 with MeOH during the isolation (Figure S10). Niphimycins contain 22 stereocenters in their polyketide chains. The planar structure of niphimycin was established more than 30 years ago;20,21,30 however, only partial configurations of these compounds, including the relative configurations of C-21/C-23/C-25/C-27/C-28 (anti/anti/anti/syn) and the absolute configurations of 34S, 35R, 36S, and 38S, have

Scheme 1. Degradation Reaction of Niphimycin Iα (5) to Yield 7 and 8

F



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Table 3. Proposed Configurations of the Niphimycin Backbone by Analysis of the Ketoreductase and Enoylreductase Core Regions in Comparison to the Assignment by Spectroscopic and Chemical Methods

predicted configurations by bioinformatics analysis domain

type of KR

Keatinge-Clay “RS” systemb

classical RS systemc

configurations assigned by spectroscopic/chemical analysis

KR2/DH2/ER1(ER3) KR3/DH3/ER3 KR4 KR7 KR8 KR9 KR10 KR11 KR12 KR14 KR16 KR17 KR18 KR20a

B1 B1 B1 B1 A1 B1 A1 B1 A1 B1 A1 B1 B1 B1

“S” (C-38) “S” (C-36) “R,R” (C-34,35) “R,R” (C-28,29) “S” (C-27) “R” (C-25) “S” (C-23) “R” (C-21) “S” (C-19) “R,R” (C-14,15) “R,S” (C-10,11) “R” (C-9) “R,R” (C-6,7) “R,R” (C-2,3)

38S 36S 34S,35R 28S,29R 27R 25S 23S 21R 19S 14S,15R 10R,11S 9S 6S,7R 2R,3R

38Sd20 36Sd20 34S,35Rd24 anti (C-28/29)e syn (C-27/28)d31 anti (C-25/27)d31 anti (C-23/25)d31 anti (C-21/23)d31 anti (C-19/21)e anti (C-14/15)e syn (C-10/11)e anti (C-9/10)e anti/syn (C-6/7/9)e anti (C-2/3)e

a Module 2 lacks an ER domain. The function is supposed to provide an ER in the neighboring module. bThe configurations labeled with quotation marks around R and S denote a deviation from the classical RS system: when assigning the configuration at the β-position of the growing polyketide chain, the lowest priority is given to the γ-carbon after the hydrogen; when discussing chirality at the α-position, the α-substituent is given the lowest priority after the hydrogen. In both cases, the carboxyl end of the chain is given the highest priority.39 cThe configurations were assigned using the Cahn−Ingold−Prelog system. dConfigurations previously established in the literature by chemical degradation and derivatization. eRelative configuration assigned in this study by spectroscopic analysis.

analysis and the relative configurational assignments obtained from NMR spectroscopic analyses, we propose the absolute configuration of all of the stereocenters of niphimycin C (3) as 2R,3R,4E,6S,7R,9S,10R,11S,14S,15R,17S,18R,19S,21R,23R,25S,27R,28S,29R,30E,32E,34S,35R,36S,38S,42E. Biological Activities. In order to obtain deeper insights into the antimicrobial properties of niphimycins, we carried out a comprehensive assay of all isolated natural niphimycin analogues against a range of Gram-positive and Gram-negative bacteria (Table S4), including the representative members of “ESKAPE” pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species).48 Compounds 1 and 5 with a single malonyl substituent showed good antimicrobial activity against methicillin-resistant S. epidermidis (MRSE) and S. aureus (MRSA), with minimum inhibitory concentrations (MICs) of 4−32 μg/mL, and modest activity against vancomycin-resistant E. faecalis and E. faecium (VRE), with an MIC of 64 μg/mL. The similar potency of 1 and 5 indicates that the position of the malonyl substituent did not exert much influence on the antibacterial activity. In contrast, the dimalonyl derivatives (2, 3, and 6) showed about 2-fold less activity than 1 and 5. These results suggest that more malonyl groups leads to decreased activity. Compound 4 was as active as 1 and 5, indicating that the antibacterial activity was not significantly affected by the ketalization of the hemiketal ring. Finally, the cleavage of the lactone of 7 to 8 led to a 2−4-fold decreased activity. Compounds 1−8 also exhibited activity against C. albicans, with MIC values of 8−32 μg/mL. All compounds were inactive against Gram-negative bacteria (MIC > 64 μg/mL).

dictating the configurations of C-34/35, C-28/29, C-25, C-21, C-14/15, C-9, C-6/7, and C-2/3, respectively, were classified as the B1 type on the basis of the presence of the conserved LxD motif and the absence of the YxP motif in the core region (Figure S2), hence leading to the “R,R”-configured α,β-stereocenters (Table 3; the quotation marks denote a deviation from the classical RS system defined by Keatinge-Clay et al., in which the lowest priority is given to the γ-carbon of the growing polyketide chain).39,45 The remaining KR domains in modules 8, 10, 12, and 16, which dictate the respective configurations of C-27, C-23, C-19, and C-10/11, were assigned to the A1-type group in accordance with the Keatinge-Clay model,39 resulting in α,β-stereocenters with “(R,)S”-configurations. The absolute configurations, which were predicted by KR domain analysis, agreed with the results of our analysis of spectroscopic data and the configurations previously established for 34S, 35R, C-21/C-23/C-25/C-27/C-28 (anti/anti/anti/syn) by chemical derivatization.24,31 A genetic analysis of ER domains by Leadlay et al.46,47 revealed that several crucial amino acid residues appear to play a critical role in the stereocontrol of the reduction process responsible for the formation of the isolated methyl-bearing center. According to their model, the presence of a tyrosine (Tyr) residue at position 52 leads to an “S”-configured center, whereas absence of Tyr52 results in an “R”-configuration. The configurations of C-36 and C-38 are determined by ER-catalyzed reduction. During niphimycin biosynthesis, all ER domains are expected to produce “S”-configured methylbearing stereocenters, as they possess the pivotal Tyr52 residue (Figure S4); this is consistent with the 36S, 38S configuration previously established by chemical degradation.20 This in turn confirmed the validity of the genetic analysis for the assignment of the absolute configuration. Therefore, based on genetic G



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secondary metabolites were detected and analyzed by AntiSMASH 4.0.14 The npm cluster boundaries that had been predicted by antiSMASH 4.0 were manually refined on the basis of functional annotation, gene distances, and comparison with the azl gene cluster. Gene function was assigned by the online BLAST program (http://blast.ncbi.nlm.nih. gov/) and Conserved Domain Database (CDD) at the NCBI server;53 modules and domains of the polyketide synthase were assigned by CDD and antiSMASH search output. The annotated sequence of the niphimycin biosynthetic gene cluster has been deposited in GenBank under the accession number MF671979. Producing Microorganism, Large-Scale Fermentation. Strain IMB7-145 was isolated from a marine sediment sample collected at a depth of ca. 40 m from Heishijiao Bay, Dalian, China. The strain was identified as a member of the genus Streptomyces based on the 16S rRNA gene sequence analysis (GenBank accession number JQ782979).13 The strain was deposited in the National Laboratory for Screening Microbial Drug, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing, China. Large-scale fermentation of strain IMB7-145 was performed using a two-stage fermentation process. The spores grown on ISP4 medium agar plates for 10 days at 28 °C were inoculated into 250 mL Fernbach flasks each containing 50 mL of the M7 medium (composed of 2.5 g of starch, 5 g of glucose, 3 g of peptone, 3 g of beef extract, 2.5 g of CaCO3, 0.001 g of FeSO4, 0.001 g of MnCl2, 0.001 g of ZnSO4, 0.001 g of CuSO4, and 0.001 g of CoCl2 in 1 L of H2O). After incubation for 2 days at 28 °C on a rotary shaker at 200 rpm, 5 mL of the seed cultures was transferred into 500 mL flasks each containing 100 mL of the M7 medium. The cultures were grown at 28 °C for 5 days at 200 rpm. Isolation of Niphimycins. The fermentation cultures (10 L) were separated into the mycelia and the supernatant by centrifugation. The resulting supernatant was extracted with XAD 7HP macroporous resin (100 g/L). The resin was thoroughly washed with H2O and then successively eluted with 50% and 100% aqueous acetone. The two latter fractions were pooled and dried under reduced pressure to produce extract A. The mycelia was extracted three times with acetone to give extract B. Extracts A and B were combined and subjected to silica gel column chromatography (CC) using gradient elutions with CH2Cl2−MeOH (20:1, 15:1, 9:1, 5:1, 3:1, 1:1, 0:1, v/v) to give 14 fractions (F1−F14). Fraction F7 was separated on Sephadex LH-20 CC eluting with 50% aqueous MeOH. The eluate was further purified on a preparative reversed-phase (RP) C18 HPLC column (Capcell AQ 5 μm, 20 mm × 250 mm, 8 mL/min), eluting with a mobile phase of MeOH−H2O (78:22) to yield 4 (12 mg) and 5 (96 mg). Fractions F8 and F9 were combined and separated by Sephadex LH-20 CC with 50% aqueous MeOH as an eluent. The eluate was further purified by RP C18 HPLC (Capcell AQ 5 μm, 20 mm × 250 mm, 8 mL/min) with an isocratic solvent of CH3CN−H2O (48:52) containing 5 mM NaH2PO4 (pH 5.0) to afford the purified fractions containing 1, 2, 3, and 6, respectively. Each fraction was diluted 5-fold with H2O and then desalted by RP C-18 solid-phase extraction to give the purified compounds, being finally lyophilized. Yields obtained were 14 mg for 1, 11 mg for 2, 9 mg for 3, and 25 mg for 6. Niphimycin C (1): white, amorphous powder; [α]20D +93.9 (c 0.70, MeOH); UV (MeOH) λmax (log ε) 192.5 (4.55), 232 (4.48) nm; ECD (4.4 × 10−4 M, MeOH) λmax (Δε) 196 (−1.15), 231 (+14.94) nm; IR νmax 3354, 2966, 2934, 1715, 1647, 1595, 1458, 1416, 1379, 1291, 1184, 1149, 1065, 991, 918, 845, 806, 690, 596 cm−1; 1H NMR (CD3OD, 500 MHz) data, Table 1; 13C NMR (CD3OD, 125 MHz) data, Table 2; HRESIMS m/z 1142.7366 [M + H]+ (calcd for C59H104N3O18, 1142.7309) and 1164.7180 [M + Na]+ (calcd for C59H103N3O18Na, 1164.7129). Niphimycin D (2): white, amorphous powder; [α]20D +67.4 (c 0.58, MeOH); UV (MeOH) λmax (log ε) 193.5 (4.37), 231.5 (4.24) nm; ECD (4.1 × 10−4 M, MeOH) λmax (Δε) 196 (−2.41), 230 (+7.52) nm; IR νmax 3358, 2958, 2930, 2854, 1713, 1674, 1458, 1415, 1380, 1318, 1202, 1250, 1139, 1092, 991, 908, 840, 802, 722 cm−1; 1H NMR (CD3OD, 600 MHz) data, Table 1; 13C NMR (CD3OD, 150 MHz) data, Table 2; HRESIMS m/z 1228.7354 [M + H]+ (calcd for C62H106N3O21, 1228.7313) and 1250.7174 [M + Na]+ (calcd for C62H105N3O21Na, 1250.7133).

We further evaluated the in vitro antibacterial activity of 1 and 5 against Mycobacterium tuberculosis (Table S5). The H37Rv strain was susceptible to both 1 and 5 at an MIC of 4 μg/mL. In addition, compounds 1 and 5 were found to be active against several M. tuberculosis clinical isolates, with MICs of 4−16 μg/mL, including one strain that is resistant to isoniazid and rifampicin. The in vitro cytotoxicity assay revealed that compounds 1 and 3−6 exhibited significant cytotoxicity against the human cervical carcinoma cell line (HeLa) with IC50 values of 3.0− 9.0 μM (Table S6), while compounds 2, 7, and 8 were inactive (IC50 > 10 μM). In summary, we identified and characterized the niphimycin gene cluster (npm) from Streptomyces sp. IMB7-145 by genome sequencing and bioinformatics analysis. Analysis of the npm gene cluster allowed us to establish a model for niphimycin biosynthesis. Guided by genomic analysis, we successfully isolated and identified six niphimycins, including four new analogues, with significant antimicrobial and cytotoxic activity. The present study is the first to propose the full absolute configuration of the niphimycins based on a combination of bioinformatic and spectroscopic analyses. The present approach involved gene cluster analyses of the ketoreductase domains for assignment of α-branching methyls and β-hydroxyls and enoylreductase domains for assignment of the isolated methylbearing stereocenters. The configurations proposed by genetic analysis were consistent with the relative configuration assigned by spectroscopic analyses and the absolute configurations previously established by chemical degradation and derivatization. This study demonstrates that the combination of chemical and bioinformatics analyses represents a powerful and highly reliable approach for the discovery of microbial natural products and stereochemical characterization of complex natural products.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer model 343 polarimeter. UV and electronic circular dichroism (ECD) spectra were recorded on a JASCO-815 CD spectrometer. IR spectra were recorded on a Nicolet 5700 FT-IR microscope spectrometer (FT-IR microscope transmission). 1D and 2D NMR spectra were obtained at 500 and 600 MHz for 1H and 125 and 150 MHz for 13C, respectively, on Bruker AVANCE III HD 500 and 600 MHz spectrometers in CD3OD, using tetramethylsilane as internal reference. HRESIMS data were measured using a Thermo LTQ Orbitrap XL mass spectrometer. Flash chromatography was performed on an Ez Purifier (Suzhou Lisure Science Co., Ltd.). Analytical HPLC was carried out using an Agilent 1200 HPLC system with a quaternary pump, an autosampler, and a diode array detector. LC-MS was performed under the same conditions on an Agilent Technologies 1100 series MSD spectrometer using an electrospray source in the positive ionization mode. Preparative HPLC analysis was carried out on a Shimadzu HPLC apparatus consisting of an LC-20AP pump and an SPD-M20A diode array detector (Shimadzu Co.). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 7% H2SO4 in 95% aqueous EtOH followed by heating. Genome Sequencing and Bioinformatics Analysis. Genomic DNA of S. sp. IMB7-145 was isolated according to the salting out protocol of the Streptomyces manual.49 Genomic DNA sequencing was performed at the Beijing Genomics Institute by using an Illumina HiSeq 4000 sequencing platform combined with a Pacific Biosciences (Pacbio) RSII platform. Approximately 1268 MB HiSeq and 1298 Pacbio clean data were generated. All of the raw data were assembled by using a combination of SMRT analysis (v2.3.0), SOAPsnp, Celera Assembler, and SSPACE-LongRead software,25,50−52 leading to approximately 11.2 Mb of contiguous sequence plus one large (ca. 162 Kb) plasmid. Putative gene clusters for the biosynthesis of H



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Niphimycin E (3): white, amorphous powder; [α]20D +87.8 (c 0.82, MeOH); UV (MeOH) λmax (log ε) 192.5 (4.55), 232 (4.43) nm; ECD (4.1 × 10−4 M, MeOH) λmax (Δε) 196 (−2.60), 231 (+10.77) nm; IR νmax 3332, 2966, 2935, 1717, 1643, 1597, 1458, 1416, 1380, 1295, 1250, 1138, 1092, 1066, 986, 918, 851, 691 cm−1; 1H NMR (CD3OD, 600 MHz) data, Table 1; 13C NMR (CD3OD, 150 MHz) data, Table 2; HRESIMS m/z 1228.7374 [M + H] + (calcd for C62H106N3O21, 1228.7313) and 1250.7194 [M + Na]+ (calcd for C62H105N3O21Na, 1250.7133). 17-O-Methylniphimycin (4): white, amorphous powder; [α]20D +77.8 (c 0.64, MeOH); UV (MeOH) λmax (log ε) 193 (4.61), 232 (4.49) nm; ECD (4.3 × 10−4 M, MeOH) λmax (Δε) 196 (−5.00), 231 (+11.07) nm; IR νmax 3325, 2932, 1718, 1642, 1560, 1458, 1412, 1379, 1292, 1252, 1137, 1092, 985, 856 cm−1; 1H NMR (CD3OD, 600 MHz) data, Table 1; 13C NMR (CD3OD, 150 MHz) data, Table 2; HRESIMS m/z 1156.7474 [M + H]+ (calcd for C60H106N3O18, 1156.7466) and 1178.7290 [M + Na]+ (calcd for C60H105N3O18Na, 1178.7285). Niphimycin Iα (5): [α]20D +94.3 (c 0.92, MeOH); ECD (4.4 × 10−4 M, MeOH) λmax (Δε) 197 (−2.27), 231 (+14.74) nm. 19-O-Malonylniphimycin (6): [α]20D +95.0 (c 0.72, MeOH); ECD (4.1 × 10−4 M, MeOH) 196 (−2.83), 230 (+10.00) nm. Reduction and Demalonylation of Niphimycin Iα (5) to Yield 7. To a solution of niphimycin Iα (5, 35 mg) in MeOH (2 mL) was added NaBH4 (4 mg), and the solution was stirred at 40 °C for 6 h. After addition of 5% aqueous acetic acid (700 μL) and evaporation of the organic solvent, the residue was subjected to RP C18 flash chromatography eluting with 5% aqueous MeOH and then MeOH. The fraction that eluted with MeOH was evaporated to afford a residue (33 mg). The residue was dissolved in 2.5% NaOMe in MeOH and stirred at room temperature for 10 h. The reaction mixture was neutralized with 1 N aqueous HCl and then purified by RP C18 HPLC using MeOH−H2O (7:3) containing 0.1% trifluoroacetic acid to produce compound 7 (22 mg). Compound 7: 1H NMR data (CD3OD, 400 MHz) δ 6.19 (dd, J = 15.2, 10.0 Hz, H-31), 6.06 (dd, J = 15.2, 10.0 Hz, H-32), 5.69 (dd, J = 15.6, 8.0 Hz, H-5), 5.65 (dd, J = 15.2, 6.4 Hz, H-30), 5.54 (dd, J = 15.2, 8.8 Hz, H-33), 5.45 (dt, J = 15.2, 6.8 Hz, H-42), 5.44 (dt, J = 15.2, 6.8 Hz, H-43), 5.42 (dd, J = 15.2, 8.0, H-4), 4.75 (dd, J = 8.0, 3.0, H-35), 3.97−4.15 (m, 7H), 3.85−3.92 (m, 2H), 3.65−3.80 (m, 4H), 3.16 (t, J = 7.2 Hz, H2-46), 2.84 (s, H3-56), 2.54 (m, H-34), 2.44 (m, H-2), 2.29 (m, H-6), 2.07 (q, J = 6.8 Hz, H2-44), 1.97 (q, J = 6.8 Hz, H2-41), 1.30−1.90 (m, 29H), 1.08 (d, J = 6.4 Hz, H3-48), 1.02 (d, J = 7.2 Hz, H3-47), 1.01 (d, J = 6.8 Hz, H3-52), 0.95 (d, J = 6.8 Hz, H3-53), 0.90 (d, J = 7.2 Hz, H3-49 and H3-50), 0.87 (d, J = 6.8, H3-51 and H3-54); 13C NMR data (CD3OD, 100 MHz), Table S3; ESI-LC-MS, Capcell AQ 4.6 mm × 150 mm, tR = 10.1 min (MeCN−5 mM NH4Ac in H2O, 30% to 70% in 25 min, 1 mL/min); UV (DAD) 232 nm; LRMS m/z 1058 [M + H]+. Cleavage of the Lactone Ring of 7 to Yield 8. Compound 7 (22 mg) was dissolved in 5% NaOMe in 5 mL of MeOH and stirred at 60 °C for 24 h. The reaction mixture was neutralized with 1 N HCl and was purified by RP C18 flash chromatography using MeOH−H2O (7:3) to yield 8 (15 mg). Compound 8: 1H NMR data (CD3OD, 400 MHz) δ 6.21 (dd, J = 15.2, 10.4 Hz, H-31), 6.09 (dd, J = 15.2, 10.4 Hz, H-32), 5.71 (dd, J = 14.8, 8.4 Hz, H-5), 5.61 (dd, J = 15.2, 6.4 Hz, H-30), 5.53 (dd, J = 15.2, 7.2 Hz, H-33), 5.45 (dd, J = 15.2, 7.2 Hz, H-42), 5.44 (dd, J = 15.2, 7.2, H-43), 5.40 (dd, J = 14.8, 6.4 Hz, H-4), 4.02−4.22 (m, 8H), 3.85−3.89 (m, 2H), 3.73−3.81 (m, 4H), 3.16 (t, J = 7.2 Hz, H2-46), 2.84 (s, H3-56), 2.38 (m, H-2), 2.30 (m, H-6), 2.28 (m, H-34), 2.07 (q, J = 6.4 Hz, H2-44), 1.98 (q, J = 6.4 Hz, H2-41), 1.30−1.95 (m, 29H), 1.11 (d, J = 7.2 Hz, H3-48), 1.06 (d, J = 6.8 Hz, H3-47), 1.01 (d, J = 6.4 Hz, H3-52), 0.94 (d, J = 7.2 Hz, H3-53), 0.92 (d, J = 7.2 Hz, H3-49 and H3-50), 0.87 (d, J = 6.4, H3-51 and H3-54); 13C NMR data (CD3OD, 100 MHz), Table S3; ESI-LC-MS, Capcell AQ 4.6 mm × 150 mm, tR = 6.7 min (MeCN−5 mM NH4Ac in H2O, 30% to 70% in 25 min, 1 mL/min); UV (DAD) 232 nm; LRMS m/z 1076 [M + H]+. Antibacterial Assay. The antimicrobial activities were assayed by using the agar dilution method as previously described.13 Cytotoxicity Assay. Cancer cell lines (HepG2, MCF-7, K562, and HeLa) were purchased from the American Type Culture Collection.

The cytotoxicities of the tested compounds against the above cancer cells were evaluated by the sulforhodamine B method described previously.54 Doxorubicin hydrochloride was used as a positive control.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00859. NMR data of compounds 5−8; additional tables and figures; MS, IR, and NMR spectra of compounds 1−4, 7, and 8 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-10-63165277. E-mail: [email protected] (M. Gan). ORCID

Maoluo Gan: 0000-0002-3089-8654 Author Contributions §

Y. Hu and M. Wang contributed equally.

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 (Grant No. 81273414), CAMS Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-2002), the Chinese National S&T Special Project on Major New Drug Innovation (Grant Nos. 2017ZX09101003-007-002 and 2015ZX09304006-016), and PUMC Youth Fund (3332016062). We thank Prof. Y. Sun of Wuhan University for helpful discussions.

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REFERENCES

(1) Bérdy, J. J. Antibiot. 2005, 58, 1−26. (2) Cooper, M. A.; Shlaes, D. Nature 2011, 472, 32. (3) Manivasagan, P.; Venkatesan, J.; Sivakumar, K.; Kim, S.-K. Microbiol. Res. 2014, 169, 262−278. (4) Subramani, R.; Aalbersberg, W. Microbiol. Res. 2012, 167, 571− 580. (5) Winter, J. M.; Behnken, S.; Hertweck, C. Curr. Opin. Chem. Biol. 2011, 15, 22−31. (6) Challis, G. L. Microbiology 2008, 154, 1555−1569. (7) Laureti, L.; Song, L.; Huang, S.; Corre, C.; Leblond, P.; Challis, G. L.; Aigle, B. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 6258−6263. (8) Schulze, C. J.; Donia, M. S.; Siqueira-Neto, J. L.; Ray, D.; Raskatov, J. A.; Green, R. E.; McKerrow, J. H.; Fischbach, M. A.; Linington, R. G. ACS Chem. Biol. 2015, 10, 2373−2381. (9) Liu, X.; Biswas, S.; Berg, M. G.; Antapli, C. M.; Xie, F.; Wang, Q.; Tang, M.-C.; Tang, G.-L.; Zhang, L.; Dreyfuss, G.; Cheng, Y.-Q. J. Nat. Prod. 2013, 76, 685−693. (10) Ziemert, N.; Alanjary, M.; Weber, T. Nat. Prod. Rep. 2016, 33, 988−1005. (11) Wang, Q.; Zhang, Y.; Wang, M.; Tan, Y.; Hu, X.; He, H.; Xiao, C.; You, X.; Wang, Y.; Gan, M. Sci. Rep. 2017, 7, 3591. (12) Tan, Y.; Hu, Y.; Wang, Q.; Zhou, H.; Wang, Y.; Gan, M. RSC Adv. 2016, 6, 91773−91778. (13) Wu, C.; Tan, Y.; Gan, M.; Wang, Y.; Guan, Y.; Hu, X.; Zhou, H.; Shang, X.; You, X.; Yang, Z.; Xiao, C. J. Nat. Prod. 2013, 76, 2153− 2157. (14) Weber, T.; Blin, K.; Duddela, S.; Krug, D.; Kim, H. U.; Bruccoleri, R.; Lee, S. Y.; Fischbach, M. A.; Müller, R.; Wohlleben, W.; Breitling, R.; Takano, E.; Medema, M. H. Nucleic Acids Res. 2015, 43, W237−243.

I



Article

(48) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.; Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Clin. Infect. Dis. 2009, 48, 1−12. (49) Kieser, T.; Bibb, M. J.; Buttner, M. J.; Chater, K. F.; Hopwood, D. A.; John Innes, F. Practical Streptomyces genetics. John Innes Foundation: Norwich; John Innes Foundation: Norwich, 2000. (50) Boetzer, M.; Pirovano, W. BMC Bioinf. 2014, 15, 211. (51) Myers, E. W.; Sutton, G. G.; Delcher, A. L.; Dew, I. M.; Fasulo, D. P.; Flanigan, M. J.; Kravitz, S. A.; Mobarry, C. M.; Reinert, K. H.; Remington, K. A.; Anson, E. L.; Bolanos, R. A.; Chou, H. H.; Jordan, C. M.; Halpern, A. L.; Lonardi, S.; Beasley, E. M.; Brandon, R. C.; Chen, L.; Dunn, P. J.; Lai, Z.; Liang, Y.; Nusskern, D. R.; Zhan, M.; Zhang, Q.; Zheng, X.; Rubin, G. M.; Adams, M. D.; Venter, J. C. Science 2000, 287, 2196−2204. (52) Li, R.; Li, Y.; Fang, X.; Yang, H.; Wang, J.; Kristiansen, K.; Wang, J. Genome Res. 2009, 19, 1124−1132. (53) Marchler-Bauer, A.; Derbyshire, M. K.; Gonzales, N. R.; Lu, S.; Chitsaz, F.; Geer, L. Y.; Geer, R. C.; He, J.; Gwadz, M.; Hurwitz, D. I.; Lanczycki, C. J.; Lu, F.; Marchler, G. H.; Song, J. S.; Thanki, N.; Wang, Z.; Yamashita, R. A.; Zhang, D.; Zheng, C.; Bryant, S. H. Nucleic Acids Res. 2015, 43, D222−226. (54) Gan, M.; Liu, B.; Tan, Y.; Wang, Q.; Zhou, H.; He, H.; Ping, Y.; Yang, Z.; Wang, Y.; Xiao, C. J. Nat. Prod. 2015, 78, 2260−2265.

(15) Harvey, B. M.; Mironenko, T.; Sun, Y.; Hong, H.; Deng, Z.; Leadlay, P. F.; Weissman, K. J.; Haydock, S. F. Chem. Biol. 2007, 14, 703−714. (16) Haydock, S. F.; Mironenko, T.; Ghoorahoo, H. I.; Leadlay, P. F. J. Biotechnol. 2004, 113, 55−68. (17) Hong, H.; Fill, T.; Leadlay, P. F. Angew. Chem., Int. Ed. 2013, 52, 13096−13099. (18) Hong, H.; Samborskyy, M.; Lindner, F.; Leadlay, P. F. Angew. Chem., Int. Ed. 2016, 55, 1118−1123. (19) Xu, W.; Zhai, G.; Liu, Y.; Li, Y.; Shi, Y.; Hong, K.; Hong, H.; Leadlay, P. F.; Deng, Z.; Sun, Y. Angew. Chem., Int. Ed. 2017, 56, 5503−5506. (20) Bassi, L.; Joos, B.; Gassmann, P.; Kaiser, H.-P.; Leuenberger, H.; Keller-Schierlein, W. Helv. Chim. Acta 1983, 66, 92−117. (21) Keller-Schierlein, W.; Joos, B.; Kaiser, H.-P.; Gassmann, P. Helv. Chim. Acta 1983, 66, 226−258. (22) Gassmann, P.; Hagmann, L.; Keller-Schierlein, W.; Samain, D. Helv. Chim. Acta 1984, 67, 696−705. (23) Ivanova, V.; Gushterova, A. J. Antibiot. 1997, 50, 965−969. (24) Laskowski, T.; Rafiński, Z.; Sowiński, P.; Pawlak, J. Magn. Reson. Chem. 2012, 50, 347−349. (25) Rhoads, A.; Au, K. F. Genomics, Proteomics Bioinf. 2015, 13, 278−289. (26) Hong, H.; Sun, Y.; Zhou, Y.; Stephens, E.; Samborskyy, M.; Leadlay, P. F. Beilstein J. Org. Chem. 2016, 12, 2164−2172. (27) Aparicio, J. F.; Molnár, I.; Schwecke, T.; König, A.; Haydock, S. F.; Ee Khaw, L.; Staunton, J.; Leadlay, P. F. Gene 1996, 169, 9−16. (28) Yurkovich, M. E.; Tyrakis, P. A.; Hong, H.; Sun, Y.; Samborskyy, M.; Kamiya, K.; Leadlay, P. F. ChemBioChem 2012, 13, 66−71. (29) Grabley, S.; Hammann, P.; Raether, W.; Wink, J.; Zeeck, A. J. Antibiot. 1990, 43, 639−647. (30) Samain, D.; Cook, J. C.; Rinehart, K. L. J. Am. Chem. Soc. 1982, 104, 4129−4141. (31) Hoyer, F.; Habermehl, G.; Duddeck, H.; Tóth, G. Magn. Reson. Chem. 1999, 37, 371−375. (32) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.; Tachibana, K. J. Org. Chem. 1999, 64, 866−876. (33) Benowitz, A. B.; Fidanze, S.; Small, P. L. C.; Kishi, Y. J. Am. Chem. Soc. 2001, 123, 5128−5129. (34) Kobayashi, Y.; Tan, C.-H.; Kishi, Y. J. Am. Chem. Soc. 2001, 123, 2076−2078. (35) Takada, K.; Imae, Y.; Ise, Y.; Ohtsuka, S.; Ito, A.; Okada, S.; Yoshida, M.; Matsunaga, S. J. Nat. Prod. 2016, 79, 2384−2390. (36) Hertweck, C. Angew. Chem., Int. Ed. 2009, 48, 4688−4716. (37) Caffrey, P. ChemBioChem 2003, 4, 654−657. (38) Reid, R.; Piagentini, M.; Rodriguez, E.; Ashley, G.; Viswanathan, N.; Carney, J.; Santi, D. V.; Hutchinson, C. R.; McDaniel, R. Biochemistry 2003, 42, 72−79. (39) Keatinge-Clay, A. T. Chem. Biol. 2007, 14, 898−908. (40) Jahns, C.; Hoffmann, T.; Müller, S.; Gerth, K.; Washausen, P.; Höfle, G.; Reichenbach, H.; Kalesse, M.; Müller, R. Angew. Chem., Int. Ed. 2012, 51, 5239−5243. (41) Ishida, K.; Lincke, T.; Hertweck, C. Angew. Chem., Int. Ed. 2012, 51, 5470−5474. (42) Essig, S.; Bretzke, S.; Müller, R.; Menche, D. J. Am. Chem. Soc. 2012, 134, 19362−19365. (43) Menche, D.; Arikan, F.; Perlova, O.; Hortsmann, N.; Ahlbrecht, W.; Wenzel, S. C.; Jansen, R.; Irschik, H.; Muller, R. J. Am. Chem. Soc. 2008, 130, 14234−14243. (44) Hartmann, O.; Kalesse, M. Angew. Chem., Int. Ed. 2014, 53, 7335−7338. (45) Keatinge-Clay, A. T.; Stroud, R. M. Structure 2006, 14, 737− 748. (46) Kwan, D. H.; Sun, Y.; Schulz, F.; Hong, H.; Popovic, B.; SimStark, J. C. C.; Haydock, S. F.; Leadlay, P. F. Chem. Biol. 2008, 15, 1231−1240. (47) Kwan, D. H.; Leadlay, P. F. ACS Chem. Biol. 2010, 5, 829−838. J


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