Note pubs.acs.org/jnp
6‑Deoxy-13-hydroxy-8,11-dione-dihydrogranaticin B, an Intermediate in Granaticin Biosynthesis, from Streptomyces sp. CPCC 200532 Bingya Jiang, Shufen Li, Wei Zhao, Ting Li, Lijie Zuo, Yanni Nan, Linzhuan Wu,* Hongyu Liu, Liyan Yu, Guangzhi Shan, and Limin Zuo Key Laboratory of Biotechnology of Antibiotics of Ministry of Health, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *
ABSTRACT: A new granaticin analogue and its hydrolysis product were isolated from Streptomyces sp. CPCC 200532. Their structures were determined to be 6-deoxy-13-hydroxy8,11-dione-dihydrogranaticins B (1) and A (2), respectively, by detailed analysis of spectroscopic data. Compound 1 was regarded as an intermediate in granaticin biosynthesis, as it was bioconvertable to granaticin B. Compared to granaticin B, 1 showed similar cytotoxicity against cancer cell line HCT116, but decreased cytotoxicity against cancer cell lines A549, HeLa, and HepG2. Compound 2 displayed lower cytotoxicity than 1 against all four cancer cell lines tested.
G
appeared (for granaticin A, Rf 0.45; for granaticin B, Rf 0.34; Figure S3). The band was scrapped off and extracted with EtOAc for LC-ESI(−)HRMS analysis. Two LC peaks with identical UV and visible spectra showed up at 8.915 and 14.574 min. The 8.915 min peak revealed a molecular ion at m/z 429.11734 ([M − H]−), and the 14.574 min peak a molecular ion at m/z 543.18519 ([M − H]−), in the hyphenated ESI(−)HRMS (Figure S4), which established molecular formulas of both compounds, as C 22 H 22 O 9 (calcd at 429.11801 for [M − H]−) and as C28H32O11 (calcd at 543.18609 for [M − H]−). Samples of the two LC peaks were collected and analyzed using TLC. The 14.574 min peak sample migrated with an Rf 0.16, and the 8.915 min peak sample had Rf 0.25 on silica gel TLC (Figure S3). Granaticin B loses the α-L-rhodinose moiety to become granaticin A upon alkaline or acid treatment. The above 8.915 min peak appeared when acetic acid was added into the mobile phase of LC. Meanwhile, the molecular formula difference of the above two compounds could be explained by the presence or absence of α-L-rhodinose (C6H10O2) in granaticin B, indicating the two compounds may be granaticin analogues. In this case, 1 would be a new granaticin analogue, as it showed a different molecular formula from any reported granaticin. A total volume of 5 L of ISP2 culture of Streptomyces sp. CPCC 200532 incubated at 28 °C for 7 d was used to isolate the two granaticin analogues. After combined purification procedures of EtOAc extraction (2.0 g), preparative TLC (73.2
ranaticin (granaticin A or B, dihydrogranaticin A or B) produced by certain Streptomyces strains is a benzoisochromanequinone (BIQ) antibiotic with antibacterial and anticancer activities.1−3 As a typical aromatic polyketide, granaticin is biosynthesized by type II polyketide synthases (PKS) with the condensation of eight two-carbon units to generate a linear octaketide, and this is followed by modification by post-PKS tailoring enzymes involving cyclization/aromatization, oxidation, and glycosylation to finally form granaticin A or B.4−6 The gene cluster for granaticin biosynthesis was cloned, and a pathway for granaticin biosynthesis was proposed.6−9 Although more than a dozen granaticins and related compounds have been identified from various granaticin producers/mutants, some details of granaticin biosynthesis such as the modification order of C-8 oxidation (or hydroxylation) and C-glycosylation were neither clear nor proved experimentally.2,3,10,11 In our chemical screening for new secondary metabolites with antibacterial or anticancer activities, we characterized Streptomyces sp. CPCC 200532, a strain from the China Pharmaceutical Culture Collection, as a granaticin producer (Figures S1 and S2). Silica gel TLC and LC-ESI(−) HRMS suggested that the strain also produced granaticin analogues. We herein report the discovery and identification of 6-deoxy13-hydroxy-8,11-dione-dihydrogranaticin B and its hydrolysis product 6-deoxy-13-hydroxy-8,11-dione-dihydrogranaticin A from this strain. Fresh spores of Streptomyces sp. CPCC 200532 were spread on ISP2 plates and incubated at 28 °C for 7 d. The culture was then extracted with ethyl acetate (EtOAc) overnight at room temperature. After concentration, the EtOAc extract was loaded on silica gel TLC for fractionation. A yellow band with Rf 0.16 © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 4, 2014
A
dx.doi.org/10.1021/np500138k | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 1. NMR Spectral Data of 1 and 2a
mg), and reversed-phase HPLC, two pure compounds, 1 (13.5 mg) and 2 (9.2 mg), were obtained as yellow, amorphous powders.
1 position
The NMR spectra of 1 (Figures S5−S10) exhibited strong similarities to those of dihydrogranaticin B (C28H31O12), which was one oxygen atom more than 1 in molecular formula.2,10 Signals for structural features of dihydrogranaticin B, such as the α-L-rhodinose moiety, the p-benzoquinone ring, and the dideoxyhexose moiety attached to the BIQ chromophore via two carbon−carbon bonds (C17−C9, C20−C10), were all found in the 1H and 13C NMR spectra of 1. An additional aromatic proton signal at δH 7.41 was observed in the 1H NMR spectrum of 1, and a methylene signal at δC 34.7 attributable to a C-4 pyran ring was significantly different from dihydrogranaticin B, dihydrokalafungin, or 8-hydroxydihydrokalafungin (δC at about 28 for C-4).2,10,12,13 In addition, HMBC spectroscopic data of 1 showed correlations from H-6 to C-4, C-11, C-12, C-13, and C-14, from H-15 to C-3, C-5, C-13, and C-14, and from H-16 to C-14 and C-15 (Figure 1). Therefore,
δC
1 2
172.0 40.9
3 4
64.3 34.7
5 6 7 8 9 10 11 12 13 14 15 16 17 18
144.5 120.8 113.5 179.7 147.2 141.4 191.8 130.6 159.0 136.0 68.6 19.1 62.5 35.5
19 20 21 22 1′ 2′ 3′ 4′ 5′ 6′ 19-OH 20-OH
76.4 79.2 73.4 17.2 95.4 24.4 26.3 67.1 67.8 17.6
δH (J in Hz) 2.66 2.56 4.44 3.01 2.75
dd (15.6, 4.2) dd (15.6, 7.8) m m m
7.41 s
5.12 1.56 4.99 2.60 1.62 4.05
m d (6.6) dd (3.0, 1.8) m m m
3.77 1.03 4.81 1.95 1.53 3.50 4.14 1.15
q (6.6) d (6.6) br s m; 1.20 m m; 1.86 m br s m d (6.0)
6.20 br s
2 δC 172.0 40.8 64.3 34.7 144.7 120.9 113.5 179.7 147.4 140.9 192.5 130.7 158.9 135.9 68.6 19.1 62.7 37.3 72.2 81.0 73.3 17.1
δH (J in Hz) 2.67 2.56 4.45 3.01 2.76
dd dd m dd dd
(15.6, 4.2) (15.6, 8.4) (17.4, 3.0) (17.4, 11.4)
7.43 s
5.11 1.56 4.95 2.62 1.47 3.92
q (6.6) d (6.6) dd (3.6, 2.4) m m d (7.8)
3.69 q (6.0) 1.02 d (6.0)
6.21 br s 4.58 br s
a1
H and 13C NMR spectral data (δ) were obtained at 600 and 125 MHz, respectively, on a VNS-600 spectrometer or Bruker 600 spectrometer and measured in acetone-d6 at room temperature.
completely by HSQC, COSY, and HMBC spectroscopic data (Figure 1), as indicated in Table 1. As 1 was coproduced with granaticin (granaticins B and A and granaticinic acid), the stereochemical features of 1, such as chiral C-3 and C-15, should be the same as granaticin (C-3R, C-15S). Compound 2 presumably has the same absolute configurations as 1, as it is identical to 1 except for the absence of the α-L-rhodinose. To prove 1 was an intermediate in granaticin biosynthesis, bioconversion of 1 to granaticin B was conducted in DMR1, a genetically engineered mutant of the granaticin producer Streptomyces vietnamensis GIMV4.0001.14 The mutant has all the post-PKS modification genes for granaticin biosynthesis, yet cannot produce any granaticin because of the inactivated granaticin biosynthesis PKS gene. Although the majority of 1 was unchanged in the bioconversion, granaticin B did appear (Figures S17 and S18), and 1 can act as an intermediate in granaticin biosynthesis. In the DMR1 bioconversion of 2, only traces of granaticin B were detected, and the majority of 2 was changed to an unidentified yellow compound with m/z 408 (Figures S17 and S19). The conversion of 1 to granaticin B provided biological evidence for C-8 oxidation and C-
Figure 1. Key COSY (bold) and HMBC (arrow) correlations of 1 and 2.
the structure of 1 was determined as 6-deoxy-13-hydroxy-8,11dione-dihydrogranaticin B. The NMR chemical shifts of 1 were assigned completely by HSQC, COSY, and HMBC spectroscopic data (Figure 1) as indicated in Table 1. The NMR spectra of 2 (Figures S11−S16) contained all the signals found in 1 except those attributable to the α-L-rhodinose unit. In addition, compared to 1, the C-18, C-19, and C-20 of 2 were shielded by ΔδC −1.8, +4.2, and −1.8 ppm, respectively. The 1H−1H COSY correlation from 19-OH to H-19 and the HMBC correlations from H-19 to C-10 and C-21, in combination with their chemical shifts, confirmed the presence of a C-19 hydroxy group in 2. Other NMR signals of 2 resembled those of 1. Consequently, the structure of 2 was determined to be 6-deoxy-13-hydroxy-8,11-dione-dihydrogranaticin A. The NMR chemical shifts of 2 were assigned B
dx.doi.org/10.1021/np500138k | J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 2. Proposed pathway of granaticin B biosynthesis with C-8 oxidation and C-glycosylation taking place before C-6 oxidation. Extraction and Isolation. The agar culture of Streptomyces sp. CPCC 200532 was extracted three times with an equal volume of EtOAc. The EtOAc extract was concentrated under reduced pressure at room temperature, then loaded onto preparative TLC plates for fractionation with a mobile phase of CH2Cl2−EtOAc−AcOH (6:4:0.1). The yellow band with Rf 0.16 was scraped off, extracted with EtOAc, dried, and redissolved in MeOH. It was refined by preparative HPLC (Agilent ZorBax SB C18, 5 μm, 9.4 mm × 250 mm, 39% MeCN in H2O containing 0.1% AcOH, 2.0 mL/min), which yielded two pure compounds, 1 (13.5 mg) and 2 (9.2 mg). Cytotoxicity Assay. Compounds 1 and 2 were evaluated for cytotoxicity against HCT116 (colon carcinoma cell line), HeLa (cervical cancer cell line), A549 (lung cancer cell line), and HepG2 (liver hepatocellular carcinoma cell line) by the MTT assay. The assays were conducted with different concentrations of 1, 2, and granaticins B and A (ranging from 0.7 to 46.0 μM for 1, 5.8 to 372.1 μM for 2, 0.6 to 35.8 μM for granaticin B, and 0.7 to 45.0 μM for granaticin A; dissolved in DMSO with a final concentration of 0.1%). Antibacterial Activity. Filter paper disks with different amounts of 1 (200−800 μg), 2 (200−800 μg), and granaticins B (0.5−4.0 μg) and A (0.5−4.0 μg) were put on YPD plates (medium composition: peptone 1.0%, beef extract 0.3%, yeast extract 0.3%, glucose 0.1%, NaCl 0.5%, agar 2.0%) mixed with Bacillus subtilis CMCC 63501 (test bacterium), then incubated at 37 °C for 16−18 h to compare the inhibition zones.16
glycosylation taking place before the C-6 oxidation in granaticin B biosynthesis (Figure 2). The cytotoxicities of 1 and 2 against four cancer cell lines were evaluated by the MTT assay. Compared to granaticin B, 1 showed similar cytotoxicity against cancer cell line HCT116, but decreased cytotoxicity against cancer cell lines A549, HeLa, and HepG2. Compound 2 displayed lower cytotoxicity than 1 against all four cancer cell lines tested (Table 2). Compound 2 was reported to slow cancer cell growth by inhibiting cell division cycle kinase (Cdc7).15 Table 2. Cytotoxicities of 1, 2, and Granaticins B and A against Four Cancer Cell Lines cytotoxicity (IC50, μM) compound
A549
HCT116
HeLa
HepG2
1 granaticin B 2 granaticin A
37.6 1.9 196.6 1.3
13.1 14.8 178.7 11.0
20.2 1.6 297.3 0.7
18.8 5.8 148.3 6.6
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Compound 1 exhibited about 200-fold less activity than granaticin B against Bacillus subtilis CMCC 63501 (Figure S20). Elson reported that dihydrogranaticin B showed 2−10 times less activity than granaticin B against various bacteria,1 and the absence of the C-6 hydroxy group in 1 reduces the antibacterial potency.
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ASSOCIATED CONTENT
S Supporting Information *
TLC and HPLC analysis of EtOAc extract of Streptomyces sp. CPCC 200532; MS and NMR spectra of 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.
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EXPERIMENTAL SECTION
General Experimental Procedures. The 1D and 2D NMR spectra of 1 and 2 (600 MHz for 1H and at 150 MHz for 13C) were recorded in acetone-d6 using a VNS-600 spectrometer or Bruker 600 spectrometer, with solvent peaks used as references. MS and HRMS were analyzed by LTQ XL and Orbitrap XL from Thermo Fisher Scientific. Analytical HPLC was conducted on a Shimadzu LC-20AT with a photodiode array detector (PAD). Preparative HPLC was accomplished on an Agilent HPLC system with 1200 quat pump and PAD detector. Preparative TLC was performed with high-performance silica gel preparative plates (HSGF254, from Yantai Chemical Industry Research Institute, Yantai, China). Fermentation. Fresh spores of Streptomyces sp. CPCC 200532 were spread on ISP2 plates (medium composition: yeast extract 0.4%, malt extract 1.0%, glucose 0.4%, and agar 1.5%) and incubated at 28 °C for 7 d.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: 86-10-63165283. Fax: 86-10-63017302. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors of the paper are very grateful to Prof. Honghui Zhu and Dr. Mingrong Deng from the Institute of Microbiology, Guangdong, China, for the generous use of DMR1 in the bioconversion experiment. This work was supported by National Natural Science Foundation of China (81302676), C
dx.doi.org/10.1021/np500138k | J. Nat. Prod. XXXX, XXX, XXX−XXX
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National Mega-project for Innovative Drugs (2012ZX09301002-001-016, 2012ZX09301002-003), Fundamental Research Funds for the Central Universities (2012N09), and National Infrastructure of Microbial Resources (No. NIMR-2013-3).
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