Article pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Methylsulfonylated Polyketides Produced by Neosartorya udagawae HDN13-313 via Exogenous Addition of Small Molecules Guihong Yu,† Qiuying Wang,† Shan Liu,‡ Xiaomin Zhang,† Qian Che,† Guojian Zhang,†,§ Tianjiao Zhu,†,§ Qianqun Gu,† and Dehai Li*,†,§
Downloaded via MACQUARIE UNIV on February 21, 2019 at 04:17:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ Marine Biomedical Research Institute of Qingdao, Qingdao 266003, People’s Republic of China § Laboratory for Marine Drugs and Bioproducts of Qingdao Pilot National Laboratory for Marine Science and Technology, Qingdao 266237, People’s Republic of China S Supporting Information *
ABSTRACT: Two new polyketides modified with a rare methylsulfonyl group, 3-methoxy-6-methyl-5(methylsulfonyl)benzene-1,2,4-triol (1) and neosartoryone A (2), along with a biogenetically related compound (3), were isolated from Neosartorya udagawae HDN13-313 cultivated with the DNA methyltransferase inhibitor 5-azacytidine. The methylsulfonyl group of 1 and 2 was proven to be derived from DMSO, which was used as the solvent to dissolve 5-azacytidine. This is the first report of a fungus that can achieve a sulfonylation-like modification of natural products utilizing DMSO as a sulfur source. Compound 2 showed lipid-lowering activity in vitro comparable to simvastatin.
N
present work, we report the isolation, structure elucidation, and
atural products from marine-derived fungi continue to play a pivotal role in drug discovery and have attracted broad interests of chemists and biologists. Whole-genome sequencing programs proved that fungi possess various biosynthetic gene clusters leading to novel chemical skeletons, e.g., polyketides, terpenoids, and peptides, and many special tailoring enzymes with specific catalytic functions for modification, degradation, and transformation of specific structures via hydroxylation, amination, thiolation, etc.1,2 Those biosynthetic gene clusters and specific tailoring enzymes represent huge metabolic potential leading to diverse bioactive structures.3 To tap into the fungal-stored biosynthetic potential, tools and methods such as the OSMAC strategy, epigenetic modification, and gene engineering are being constantly expanded.1 During our recent exploration of fungal strains with high secondary metabolite producing potential, we performed a pilot screening process that involves adding small-molecule epigenetic modifiers of histone deacetylase (HDAC) and DNA methyltransferase (DNMT) to fungal cultures and examining how the metabolic profiles are affected.4−6 As a result, a mangrove-derived fungus Neosartorya udagawae HDN13-3137 was found to be sensitive to the DNA methyltransferase inhibitor 5-azacytidine, which led to the appearance of new peaks in the UPLC-UV analysis whether under static or agitated fermentation conditions. During further chemical study, two new aromatic polyketides (compounds 1 and 2) with a rare methylsulfonyl modification of an aromatic ring were obtained. Compound 2 showed lipid-lowering activity in vitro. In the © XXXX American Chemical Society and American Society of Pharmacognosy
biological activities of these compounds.
■
RESULTS AND DISCUSSION The N. udagawae HDN13-313 strain was cultured under agitated and static fermentation, with the addition of 5azacytidine (1.33 mL of a 55 mg/mL solution in DMSO was added per liter of the culture medium). EtOAc extracts were fractionated by ODS MPLC and HPLC to yield compounds 1− 3. Among them, compound 1 was isolated from the agitated fermentation, while 2 and 3 were found from the static fermentation. Compound 1 was obtained as a pale yellow powder with the molecular formula C9H12O6S based on the HRESIMS ion detected at m/z 247.0283 [M − H]−. The 1D NMR resonances indicated the presence of six nonprotonated aromatic carbons (δH 145.2, 144.9, 137.6, 134.5, 119.9, 114.3), three exchangable protons (δH 9.85, 9.60, 8.32), and three methyls including a Received: January 12, 2019
A
DOI: 10.1021/acs.jnatprod.9b00035 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
methoxy (δC 60.7, δH 3.66) and a methylsulfonyl group (δC 45.6, δH 3.24). Based on the molecular formula and the chemical shifts, compound 1 possessed an aromatic ring substituted by three hydroxy groups, one methyl, one methoxy, and one methylsulfonyl group. The HMBC correlations from H3-8 to C3, from 2-OH to C-1/C-2/C-3, from H3-7 to C-1, and from H3-9 to C-1/C-5/C-6 suggested the methoxy, one of the hydroxy groups, the methylsulfonyl group, and a methyl were linked to C-3, C-2, C-1, and C-6, respectively (Figure 1). Meanwhile, the
Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Data of Compound 2 (DMSO-d6)
remaining two OH groups can be placed at C-4 and C-5 according to the chemical shifts and the molecular formula. Although the HMBC correlations concerning 4-OH and 5-OH were not detected in compound 1, we found the correlation of 4OH to C-3/C-4/C-5 and 5-OH to C-4/C-5/C-6 in the HMBC spectrum of deuterated-1 (deuterated at the methylsulfonyl group), which was obtained by incubating N. udagawae HDN13-313 with deuterated DMSO (vide infra), assigning the chemical shifts of 4-OH to δH 9.85 and 5-OH to δH 8.32, respectively. Compound 2 was obtained as a pale yellow powder with the molecular formula C17H14O8S established by the HRESIMS ion detected at m/z 379.0486 [M + H]+. The 1D NMR data of 2 indicated the presence of 10 nonprotonated carbons (including two carbonyls), four aromatic methines, one oxygenated methylene, and two methyls (including a methoxy) (Table 2).
δC, type 114.3, C 144.9, C 134.5, C 145.2, C 137.6, C 119.9, C 45.6, CH3 60.7, CH3 12.7, CH3
1 2 3 4 4a 5 5a 6 7 8
160.9, C 108.7, CH 155.7, C 104.7, CH 156.0, C 119.2, CH 134.8, C 146.9, C 120.3, CH 167.8, C
δH 6.84, s 7.07, s 8.28, s
7.97, s
no.
δC, type
8a 9 9a 10 11 12 13 1-OH 7-OH 11-OH
120.3, C 179.8, C 108.0, C 155.9, C 62.7, CH2 53.5, CH3 43.1, CH3
δH (J in Hz)
4.62, d (5.8) 3.93, s 3.44, s 11.78, s 5.57, t (5.9)
Figure 2. UPLC-MS analysis ([M − H]−: m/z 247 of 1) of standard compound and the fermentation broth extracts from N. udagawae HDN13-313 incubated with 5-azacytidine (dissolved in DMSO, 1.33 mL solution was added per liter of the culture medium), with DMSO (1.33 mL per liter of the culture medium) and no 5-azacytidine (control II), and without DMSO and 5-azacytidine (control I).
Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data of Compound 1 (DMSO-d6) no.
δC, type
In natural products, sulfur mainly exists as thiols, thioethers, sulfonamides, isothiocyanates, and sulfonic acids and only in a very few cases in the form of sulfones, such as the agelasidines, adociaquinones, and scedapin C, in which the sulfur was proposed to be derived from L-methionine, L-cysteine, or sulfite.9−14 The rare methylsulfonyl group of 1 and 2 prompted our interest in investigating their routes of formation. Careful analysis of the UPLC-MS spectra showed that compound 1 could be produced by incubating the fungus N. udagawae HDN13-313 only with DMSO under agitation, which suggested that DMSO participated in the biosynthesis of 1, while 5azacytidine was not necessary (Figure 2). To support this
Figure 1. Key COSY and HMBC correlations of 1 (dashed arrows mean signals detected from deuterated-1) and 2.
1 2 3 4 5 6 7 8 9 2-OH 4-OH 5-OH
no.
δH
opinion, we cultivated the fungus HDN13-313 with deuterated DMSO (DMSO-d6) in the culture medium. As we expected, compound 1 with a deuterated methylsulfonyl group was produced, which suggested that N. udagawae HDN13-313 could degrade and utilize DMSO in the biological synthesis (UPLCMS data and 1H NMR data, see Figure S1 and Figure S16, respectively). Different from compound 1, compounds 2 and 3 could not be detected when the fungus N. udagawae HDN13313 was cultivated only with DMSO in the medium (Figure 3). Further investigation showed that compound 3 could be produced by adding 5-azacytidine alone (dissolved in H2O), while compound 2 was only produced by adding both DMSO and 5-azacytidine, suggesting that 5-azacytidine could activate the biosynthetic genes of the dibenzoxepindiones (for example, compound 3), and then the fungus N. udagawae HDN13-313 further embedded the degradation product of DMSO in compound 2 (Figure 3). Similarly, when we cultivated fungus N. udagawae HDN13-313 with DMSO-d6 in the solid medium,
3.24, s 3.66, s 2.33, s 9.60, s 9.85, s 8.32, s
The dibenzoxepindione core of 2 was deduced by the similar 13C NMR data to reference compound 4 (this compound has not been named)8 and the HMBC correlations from H-7 to C-5/C8/C-8a, H-5 to C-6/C-9/C-10, H-2 to C-1/C-4/C-9a, and H-4 to C-3/C-4a/C-9a (Figure 1). Finally, the structure of 2 was constructed by attaching the methylsulfonyl (δC 43.1, δH 3.44 s) group to C-6 according to the HMBC correlation from H3-13 to C-6 (δC 146.9). B
DOI: 10.1021/acs.jnatprod.9b00035 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Figure 3. UPLC-MS analysis ([M − H]−: m/z 377 of 2, m/z 315 of 3) of standard compounds and the fermentation broth extracts from N. udagawae HDN13-313 incubated with 5-azacytidine (dissolved in DMSO, 1.33 mL solution was added per liter of the culture medium), with 5-azacytidine and no DMSO (H2O as the solvent), with DMSO (1.33 mL per liter of the culture medium) and no 5-azacytidine (control II), and without DMSO and 5azacytidine (control I).
compound 2 deuterated at the methylsulfonyl group was also detected, which supported our speculation (Figure S2). Literature searches revealed that most DMSO-degrading/ biotransformation microorganisms, such as Arthrobacter and Hyphomicrobium species, first reduce DMSO to dimethylsulfide (DMS), then oxidize DMS to sulfate and formaldehyde/carbon dioxide,15 while a few cases reported that strains such as Rhodococcus sp. SY1, Alcaligenes sp. E1, and Cryptococcus humicolus WU-2 first oxidize DMSO to dimethyl sulfone (DMSO2), which is further degraded to MeOH, methane, and sulfate.16 Further investigation will be needed to discern how the methylsulfonyl group is installed in N. udagawae HDN13-313. All the compounds were evaluated for their cytotoxicity (on HL-60, HCT-116, K562, and HeLa cell lines), but none of them presented a cytotoxic effect at a concentration of 30 μM. Lipidlowering effects were also evaluated against oleic acid (OA)elicited lipid accumulation in HepG2 liver cells by Oil Red O staining. Compound 2 decreased the lipid accumulation elicited by OA at a concentration of 10 μM, with an effect comparable to the positive drug simvastatin (Figure 4). In summary, two new polyketides with a rare methylsulfonyl group, along with one biogenetically related compound, were isolated from the endophytic fungus N. udagawae HDN13-313 in the presence of the DNA methyltransferase inhibitor 5azacytidine and DMSO, which existed as a solvent. The methylsulfonyl group of 1 and 2 was proved to be derived from DMSO by an isotope labeling experiment. This is the first report of a fungus that could biodegrade DMSO and attach the degradation product to an aromatic ring. In addition, compound 2 had a lipid-lowering effect, which is also the first finding on the lipid-lowering activity of dibenzoxepindione derivatives.
Figure 4. Effects of compounds on OA-elicited intracellular lipid accumulation. Cells were treated with 10 μM of indicated compounds or simvastatin (as positive control) in DMEM + 80 μM OA or with DMEM alone (as blank) or DMEM + 80 μM OA (as negative control) for 24 h. Neutral lipids were determined by spectrophotometry at 358 nm after Oil Red O staining. Bars depict the means ± SEM of at least three experiments. **P < 0.01, *P < 0.05 OA versus blank; ##P < 0.01, # P < 0.05, test group versus OA group. OA: oleic acid.
■
EXPERIMENTAL SECTION
General Experimental Procedures. UV spectra were taken on a Beckman DU 640 spectrophotometer. IR spectra were measured on a Bruker Tensor-27 spectrophotometer in KBr discs. NMR spectra were recorded on an Agilent 500 MHz DD2 spectrometer using tetramethylsilane as an internal standard. ESIMS were obtained on a Thermo Scientific LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) or Micromass Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer (Waters Corporation). Semipreparative HPLC was performed on an ODS column (YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 3 mL/min, YMC Co., Ltd.). Medium-pressure liquid chromatography (MPLC) was performed on a Bona-Agela CHEETAHTM HP100 (Beijing Agela Technologies Co., Ltd.).17 Fungal Material. As described previously.7 C
DOI: 10.1021/acs.jnatprod.9b00035 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
■
Fermentation. The Neosartorya udagawae HDN13-313 was cultured under both agitation and static fermentation, with the addition of 5-azacytidine (dissolved in DMSO with a concentration of 55 mg/ mL; 1.33 mL solution was added per liter of the culture medium). The agitated fermentation was performed in 500 mL Erlenmeyer flasks containing 150 mL of liquid medium: glucose 4.0%, peptone 1.0%, NaCl 0.5%, and H2O, incubated at 28 °C on rotary platform shakers at 180 rpm for 9 days. The static fermentation was performed in 9 cm culture dishes containing 15 mL of solid medium: glucose (20.0 g/L), potato (200.0 g/L), agar power (25.0 g/L), and seawater (Huiquan Bay, Yellow Sea), incubated for 20 days at 28 °C. Extraction. For the agitated fermentation, after 9 days’ incubation, the whole broth (1.5 L) was filtered through cheesecloth to separate the broth supernatant and mycelia. The former was extracted with EtOAc, while the latter was extracted with MeOH. The MeOH extract was evaporated under reduced pressure to afford an aqueous solution and then extracted with EtOAc. The two EtOAc extracts were combined and concentrated under reduced pressure to give an organic extract (1.0 g). For the static fermentation (2.5 L), after 20 days’ incubation, the solid medium with mycelia was extracted with MeOH. The MeOH extract was evaporated under reduced pressure to afford an aqueous solution, which was then extracted with EtOAc. The organic layer was concentrated under reduced pressure to give an extract (3.5 g). Purification. The extract (1.0 g) of the agitated fermentation was separated by MPLC using a gradient elution of MeOH−H2O, yielding eight subfractions (fractions 1−8). Fraction 4 was separated by semipreparative HPLC eluting with MeOH−H2O (35:65) to furnish compound 1 (5.0 mg, tR 21.5 min). The extract (3.5 g) of the static fermentation was separated by MPLC using a gradient elution of MeOH−H2O, yielding nine subfractions (fractions 1−9). Fractions 6 and 7 were separated by semipreparative HPLC eluting with MeOH− H2O (50:50, 60:40) to furnish compounds 3 (6.0 mg, tR 16.5 min) and 2 (5.0 mg, tR 18.0 min), respectively. 3-Methoxy-6-methyl-5-(methylsulfonyl)benzene-1,2,4-triol (1): pale yellow powder; UV (MeOH) λmax (log ε) 215 (4.05), 245 (3.06), 300 (2.01) nm; IR (KBr) νmax 3353, 3175, 1620, 1512, 1474, 1400, 1270, 1114, 995, 973, 758, 677, 569, 510 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 247.0283 [M − H]− (calcd for C9H11O6S, 247.0282). Neosartoryone A (2): pale yellow powder; UV (MeOH) λmax (log ε) 212 (4.08), 261 (3.53), 308 (1.68), 274 (1.03) nm; IR (KBr) νmax 3502, 3080, 2924, 1743, 1650, 1474, 1417, 1293, 1138, 1021, 861, 778, 528 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 379.0486 [M + H]+ (calcd for C17H15O8S, 379.0482). Cell-Based Lipid Accumulation Assay. HepG2 cells of logarithmic growth phase were seeded in a 96-well plate at a concentration of 1.2 × 104 cells/well, 100 μL/well. After 12 h, the medium was changed to serum-free DMEM medium (80 μL/well) when 70−80% confluence was reached. Then the culture was starved for 12 h, and 20 μL of serum-free DMEM medium was added to the blank control group and OA (final concentration, 80 μM) was added to the other groups, 10 μL/well. On this basis, the OA group was supplemented with 10 μL of serum-free DMEM medium, and other groups were supplemented with 10 μL of the test compound or simvastatin with the final concentration of 10 μM. After incubating for 24 h in the incubator, the medium was discarded and washed with PBS buffer (room temperature) once. Then 4% paraformaldehyde fixative was added at 80 μL/well and fixed for 0.5 h at room temperature. The plates were washed with PBS once and rinsed with 60% 2-propanol for 10 min. A 0.3% Oil Red O staining solution (Sigma O0625) was added at 60 μL/well; the contents were stained for 1 h at room temperature and then washed three times with PBS buffer. Last, the contents were dissolved by 100 μL/well DMSO, and the OD values were measured at 358 nm.18 Assay for Cytotoxicity. As previously reported.19
Article
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00035. UPLC-MS analysis of the extract obtained from culturing Neosartorya udagawae HDN13-313; NMR, HRESIMS, and IR spectra of compounds 1 and 2 (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail:
[email protected]. ORCID
Qian Che: 0000-0003-0610-1593 Dehai Li: 0000-0002-7191-2002 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was financially supported by the Scientific and Technological Innovation Project, by Qingdao National Laboratory for Marine Science and Technology (2015ASKJ02, 2016ASKJ08-02), and NSFC-Shandong Joint Fund for Marine Science Research Centers (U1606403).
■
REFERENCES
(1) Scherlach, K.; Hertweck, C. Org. Biomol. Chem. 2009, 7, 1753− 1760. (2) Sanchez, J. F.; Somoza, A. D.; Keller, N. P.; Wang, C. C. Nat. Prod. Rep. 2012, 29, 351−371. (3) Choudhary, N.; Singh, S.; Rai, J.; Inamullah; Sharma, S. Int. J. Chem. Pharm. Sci. 2013, 1, 273−280. (4) Zhang, Z.; He, X.; Wu, G.; Liu, C.; Lu, C.; Gu, Q.; Che, Q.; Zhu, T.; Zhang, G.; Li, D. J. Nat. Prod. 2018, 81, 1651−1657. (5) He, X.; Zhang, Z.; Che, Q.; Zhu, T.; Gu, Q.; Li, D. Arch. Pharmacal Res. 2018, 41, 57−63. (6) He, X.; Zhang, Z.; Chen, Y.; Che, Q.; Zhu, T.; Gu, Q.; Li, D. J. Nat. Prod. 2015, 78, 2841−2845. (7) Yu, G.; Zhou, G.; Zhu, M.; Wang, W.; Zhu, T.; Gu, Q.; Li, D. Org. Lett. 2016, 18, 244−247. (8) Carvalho, M. R.; Barbosa, L. C. D. A.; Queiróz, J. H. D.; Howarth, O. W. Tetrahedron Lett. 2001, 42, 809−811. (9) Feng, M.; Tang, B.; Liang, S. H.; Jiang, X. Curr. Top. Med. Chem. 2016, 16, 1200−1216. (10) Waldman, A. J.; Ng, T. L.; Wang, P.; Balskus, E. P. Chem. Rev. 2017, 117, 5784−5863. (11) Prinsep, M. R. Stud. Nat. Prod. Chem. 2003, 28, 617−751. (12) Morales, J. J.; Rodriguez, A. D. J. Nat. Prod. 1992, 55, 389−394. (13) Nakamura, H.; Wu, H.; Kobayashi, J.; Ohizumi, Y.; Hirata, Y.; Higashijima, T.; Miyazawa, T. Tetrahedron Lett. 1983, 24, 4105−4108. (14) Huang, L.; Xu, M.; Li, H.; Li, J.; Chen, Y.; Ma, W.; Li, Y.; Xu, J.; Yang, D.; Lan, W. Org. Lett. 2017, 19, 4888−4891. (15) Borodina, E.; Kelly, D. P.; Schumann, P.; Rainey, F. A.; WardRainey, N. L.; Wood, A. P. Arch. Microbiol. 2002, 177, 173−183. (16) Horinouchi, M.; Kasuga, K.; Nojiri, H.; Yamane, H.; Omori, T. FEMS Microbiol. Lett. 1997, 155, 99−105. (17) Yu, G.; Wang, S.; Wang, L.; Che, Q.; Zhu, T.; Zhang, G.; Gu, Q.; Guo, P.; Li, D. Mar. Drugs 2018, 16, 25. (18) Zhang, X.; Wu, C.; Wu, H.; Sheng, L.; Su, Y.; Zhang, X.; Luan, H.; Sun, G.; Sun, X.; Tian, Y.; Ji, Y.; Guo, P.; Xu, X. PLoS One 2013, 8, No. e61922. (19) Du, L.; Zhu, T.; Liu, H.; Fang, Y.; Zhu, W.; Gu, Q. J. Nat. Prod. 2008, 71, 1837−1842. D
DOI: 10.1021/acs.jnatprod.9b00035 J. Nat. Prod. XXXX, XXX, XXX−XXX
