Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Ustilobisorbicillinol A, a Cytotoxic Sorbyl-Containing Aromatic Polyketide from Ustilaginoidea virens Daowan Lai,†,§ Jiajia Meng,†,§ Xuping Zhang,† Dan Xu,† Jungui Dai,‡ and Ligang Zhou*,† †
Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, China
‡
Downloaded via MIDWESTERN UNIV on February 20, 2019 at 18:16:38 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
S Supporting Information *
ABSTRACT: Ustilobisorbicillinol A (1), which is a novel bisorbicillinoid featuring a unique cage structure that incorporates one sorbicillinol and one sorbyl-containing phenanthrenone unit, was isolated from the culture of Ustilaginoidea virens. Three biogenetically related new metabolites (2−4) were also isolated. Their structures were elucidated by extensive spectroscopic analyses, including the 13C NMR and electronic circular dichroism (ECD) calculations for the configurational assignment. The biosynthetic pathway for these sorbyl-containing polyketides was proposed. Compound 1 showed pronounced cytotoxicity, and it induced significant cell cycle arrest and apoptosis.
S
Ustilobisorbicillinol A (1) was isolated as a yellow amorphous powder, which exhibited a prominent pseudomolecular ion peak at m/z 613.2060 [M + H]+ (calcd for C35H33O10, 613.2068) in the HRESIMS spectrum, indicating a molecular formula of C35H32O10 , with 20 degrees of unsaturation. The UV spectrum showed maximum absorptions at 240, 334, 386 nm that were similar to the reported bisorbicillinols.9 Its IR spectrum revealed the presence of hydroxyl (3727, 3452 cm−1), keto group (1737, 1722 cm−1), aromatic ring and olefinic groups (1654, 1618, 1577, 1561 cm−1). The 1H NMR spectrum of 1 (Table S2 in the Supporting Information) showed signals attributable to one aromatic proton (δH 8.99, s), eight olefinic protons [δH 7.76 (d), 7.61 (dd), 7.31 (dd), 6.59 (ddd), 6.56 (d), 6.49 (dq), 6.42 (ddd), 6.33 (dq)], three methines [δH 4.32 (d), 4.96 (d), 3.63 (dd)], and five methyl groups [δH 2.82 (s), 1.93 (d), 1.90 (d), 1.55 (s), 1.31 (s)]. The 13C NMR spectrum (Table S2) displayed a total of 35 resonances, which could be assigned to 4 keto groups (δC 204.1, 198.9, 198.3, 195.3), 20 sp2 hybrid carbons (δC 173.3−106.9) including 11 quaternary ones, and 11 sp3 hybrid carbons that included 3 methines (δC 58.0, 54.7, 44.4), 5 methyl groups (δC 19.1, 19.0, 18.5, 15.7, 13.6), and 3 quaternary ones (δC 105.9, 81.0, 65.0), as revealed by HSQC experiment. Detailed analysis of the 1H−1H COSY spectrum allowed the construction of two spin systems, that spanned from H-8 (δH 6.56, d) to CH3-12 (δH 1.90, d), and from H-12′ (δH 7.76, d) to CH3-16′ (δH 1.93, d), respectively (Figure 2a). The HMBC correlations (Figure 2a) from H-8 and H-9 (δH 7.31, dd) to C-7 (δC 173.3, C), and from H-12′ and H-13′ (δH
orbicillinoids are a family of fungal metabolites that are related to the hexaketide sorbicillin, and typically contain a sorbyl side chain in the structures.1 Around 90 members have been isolated so far from a variety of fungi that belong to 12 genera.2 Many of them have highly oxygenated and complex cyclic structures and displayed various biological activities such as cytotoxic, antioxidant, and antifungal activities, thus they were attractive candidates for developing new pharmaceutical or agrochemical agents.2 The unique structural features of these molecules make them interesting targets for realizing a total synthesis, for example, the syntheses of bisorbicillinol, bisorbibutenolide, trichodimerol, and bisorbicillinolide have been reported.3 As part of our ongoing project to discover bioactive compounds from Ustilaginoidea virens (teleomorph: Villosiclava virens),4 which is a pathogenic fungus that causes the rice false smut, we analyzed the genome of U. virens,5 to mine the gene clusters for secondary metabolites by using antiSMASH.6 Four type 1 PKS gene clusters were found, among which one cluster (No. 28) (see Figure S1 and Table S1 in the Supporting Information) showed 85% similarity with the sorbicillin biosynthetic gene cluster.7 Previously, there was no report on the isolation of sorbicillinoid from the genus Ustilaginoidea. Since sorbicillinoids have intriguing structural diversity, the isolation and identification of sorbicillinoids from U. virens were performed. After comparison of different culture media, we found that U. virens was capable of producing sorbicillinoids when fermented on a solid rice medium. Chemical investigation of the fungal extract led to the isolation of the heterodimer ustilobisorbicillinol A (1), together with its biosynthetically related monomers, ustilanthracins A (2) and B (3), ustinaphthalin (4) (Figure 1), and oxosorbicillinol.8 © XXXX American Chemical Society
Received: December 23, 2018
A
DOI: 10.1021/acs.orglett.8b04101 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
198.3), as well as from the methine (H-1; δH 4.32, d) to C-2, C-3, C-5, C-6 (δC 106.9), and C-13 (δC 18.5), allowed the construction of a 3,5-cyclohexanedione ring. Further HMBC correlations from H-10 (δH 6.42, ddd) to C-6, and from 7-OH (δH 14.98) to C-6, C-7 (δC 173.3), and C-8 (δC 120.1) established the linkage of the enol-sorbyl chain (C-7−C-12) to this ring, which was consistent with the observed NOESY correlation between H-1 and H-8 (δH 6.56, d) (Figure 2a). Thus, unit I was determined as shown in Figure 2a, which was structurally related to sorbicillinol. The second unit (II) was also constructed by careful analysis of the 2D NMR spectra (Figure 2a). The elucidation was started from H-10′, an aromatic proton (δH 8.99, s) that resonated at quite a downfield region. This suggested a carbonyl group was adjacent to it. As expected, H-10′ showed HMBC correlation to the carbonyl (C-11′, δC 195.3) of the sorbyl chain (C-11′−C16′). The ortho relationship of H-10′ and this chain was confirmed by the HMBC correlation observed from H-12′ (δH 7.76, d) to C-9′ (δC 120.4), as well as by the NOESY correlation seen between H-10′ and H-12′. The chelated hydroxyl group (8′−OH, δH 13.03) was quickly assigned to the vicinal position of this sorbyl chain, as crosspeaks were seen from 8′−OH to C-7′ (δC 118.4), and C-8′ (δC 158.3), and C-9′ in the HMBC spectrum. By the same token, CH3-17′ (δH 2.82) was positioned at C-7′, by analysis of the HMBC correlations from this methyl to C-6a′ (δC 132.7), C7′, and C-8′. The HMBC correlations from H-10′ to C-8′ and 6a′ were consistent with the aforementioned deduction, thus a pentasubstituted A-ring was deduced. The HMBC correlations from the methine (H-1′, δH 4.96) to C-10a′ (δC 121.6), C-10b′ (δC 136.4), and C-4a′ (δC 116.5), as well as from H-10′ to C-10b′, indicated the C4a′ C10b′ double bond was connected to ring A via C10a′-C10b′. This was consistent with the observed NOESY correlation between H-1′ and H-10′. The HMBC correlations from the third chelated hydroxyl group (δH 12.22) to C-4a′, C-5′ (δC 145.0), and C-6′ (δC 140.1) suggested that this OH group was located at C-5′, while the remaining carbonyl group (δC 204.1, C-4′) attached to C-4a′ such that an intramolecular hydrogen bond between 5′−OH/CO-4′ could be formed. By taking into consideration of the chemical shifts of C-5′ and C-6′, C-6′ has to be oxygenated. The long-range HMBC correlation from CH3-17′ to C-6′ hinted a direct linkage between C-6′ and C6a′. Thus, a heptasubstituted naphthalene moiety consisting of rings A and B was elucidated. One methine (δC 58.0; δH 3.63) and one dioxygenated quaternary carbon (δC 105.9) were left unassigned. In the HMBC spectrum, these carbons were found to have crosspeaks with H-1′. As there was no 1H−1H COSY correlation between H-1′ and the unassigned methine, the dioxygenated quaternary carbon had to be placed between these two methines, i.e., C-2′, then the unassigned methine was located at C-3′. The HMBC correlations from H-3′ to C-4′ and C-4a′ established the linkage between C-3′ and C-4′, hence, the cyclohexenone ring (C) was constructed. This allowed the establishment of a phenanthrenonic unit (unit II). The fusion pattern of units I and II was revealed by 1H−1H COSY, HMBC, and NOESY experiments (Figure 2b). The 1 H−1H COSY correlation between H-1 and H-3′ suggested a direct linkage of C-1 and C-3′, which was confirmed by the HMBC correlations of H-3′/C-6, and H-1/C-2′. Meanwhile, the HMBC correlations from H-1′ to C-4, C-5 and C-14, and from CH3-14 to C-1′ revealed a σ bond between C-1′ and C-4.
Figure 1. Structures of 1−4.
Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations of 1, revealing (a) the structures of units I and II, and (b) their fusion pattern.
7.61, dd) to C-11′ (δC 195.3, C) confirmed the presence of one enol-sorbyl and one sorbyl side chain, suggesting a bisorbicillinoid nature of 1. The first unit (I) was easily recognized by analysis of the 2D NMR spectra (HMBC, NOESY). The HMBC correlations from the singlet methyl (CH3-13; δH 1.55) to C-1 (δC 44.4), C-2 (δC 81.0), and C-3 (δC 198.9), and from another singlet methyl (CH3-14; δH 1.31) to C-3, C-4 (δC 65.0), and C-5 (δC B
DOI: 10.1021/acs.orglett.8b04101 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
The sorbicillinoid family includes almost all the sorbylcontaining natural compounds,1 the sorbyl-containing building block of which was exclusively derived from a hexaketide, usually sorbicillinol. Although few of the hexanoyl-containing anthraquinones, such as norsolorinic acid,10 were reported, hexanoyl-, 2,3-dihydrosorbyl-, or sorbyl-fused naphthoquinone and phenanthrenone have not been reported, to the best of our knowledge. The existence of 1−4 adds a new dimension to the diversity of the sorbicillinoid family. Because of the similar building block found in the isolated sorbyl-containing, aromatic polyketides (highlighted with dash box, see Scheme S1 in the Supporting Information), it was hypothesized that these metabolites were biosynthesized in a similar manner. These sorbyl-polyketides could be biosynthesized by two PKS, with the highly reducing iterative PKS (HRiPKS) producing the sorbyl chain while the nonreducing iPKS (NR-iPKS) extending it several times to yield the final products, by analoging to the biosynthesis of sorbicillinol (Figure S1 in the Supporting Information).7,11 In U. virens, the two PKS genes, UV8b_6010 (usorB) and UV8b_6011 (usorA), located in a same cluster, showed 65%, 70% similarity to sorB and sorA in P. chrysogenum,11a respectively, while the auxiliary modifier genes UV8b_6017 and UV8b_7688 showed 58% and 36% similarity to sorC and sorD, respectively (Figure S1, and Table S1), suggesting the same biosynthetic route for sorbicillinol and oxosorbicillinol. Indeed, mutations that disrupted either the usorA or usorB gene abolished the production of any sorbicillinoid (Figures S7 and S8 in the Supporting Information). The biosynthetic pathway for the sorbyl-containing monomeric polyketides (2−4) was thus proposed (Scheme S1). It was hypothesized that UsorA could synthesize the sorbyl part, while the NR-iPKS would elongate it with one methyl and different numbers of acetate units that underwent similar reactions, including aldol condensations and Knoevenagel cyclization as key steps, to give the (multi)cyclic structures, resulting in a similar arrangement of the substituents in the sorbyl-connecting ring. Such a plasticity in the polyketides biosynthesis was striking. The heterodimer 1 was proposed to be biosynthesized from the phenanthrenedione 1p (Scheme S1) and sorbicillinol through two rounds of Michael addition and one ketalization (see Figure 4). Although only one cluster harboring two PKS-encoding genes (Figure S1) was found, it was unclear if unknown NR-
This was consistent with the NOESY correlation between H-1′ and CH3-14. By consideration of the molecular formula and the degrees of unsaturation, C-2 and C-2′ had to be connected via an oxygen bridge to complete the entire structure of 1 (Figure 2b). The relative configuration of the chiral centers was defined by analysis of the NOESY correlations and J values. The correlations of H-1/H-3′, and H-1/CH3-13 suggested that these protons were proximal in space, while the correlation between H-1′ and CH3-14 indicated they were co-oriented. A typical long-range w-type coupling was found between H-1′ and H-3′ (4J = 2.0 Hz), thus defining the relationships of the above protons, as shown in Figure 2b. With regard to the stereochemistry of 2′−OH, it should be cis to H-3′, because of the enhanced stability of a cis over a trans 5/6 ring junction, as reported previously.9 The large coupling constants of 3JH‑8/H‑9, 3 JH‑10/H‑11, 3JH‑12′/H‑13′, and 3JH‑14′/H‑15′ (14.6−15.1 Hz) inferred the E-configuration of each double bond in the side chains. Therefore, ustilobisorbicillinol A (1) was determined as a heterodimer of sorbicillinol-related monomer and an unprecedented sorbyl-containing phenanthrenone monomer. Since unit II of 1 has less proton-containing carbons except the sorbyl chain, it was a challenge for NMR-based structural analysis. We succeeded in elucidating the structure by extensive analysis of the 2D NMR spectra, especially the HMBC correlations from hydroxyl and methyl groups. Nevertheless, 13C NMR computations were performed to provide further evidence to support the proposed structure. The calculated 13C NMR data showed good agreement with the experimental values, with an average absolute deviation of 2.52 ppm (Table S3 in the Supporting Information). The absolute configuration of 1 was determined by comparison of its experimental electronic circular dichroism (ECD) spectrum with the computed one (Figure 3). The calculated ECD spectrum of (1S, 2S, 4R, 1′R, 2′S, 3′R)-1 matched the experimental spectrum, thus the absolute configuration of 1 was determined.
Figure 3. Calculated and experimental ECD spectra of 1.
Ustilanthracins A (2) and B (3) were elucidated as sorbyllinked anthraquinones. They shared the same skeleton, but differed in the carboxyl-containing side chain, where 3dioxygenated butyric acid and 2-methyl-3-oxygenated butyric acid were found in 2 and 3, respectively. Ustinaphthalin (4) was identified as 2-methoxy-6-sorbyl-7-hydroxyl-8-methyl-1,4naphthoquinone (for the detailed structure elucidation, see the Supporting Information).
Figure 4. Proposed biosynthetic pathway of 1. C
DOI: 10.1021/acs.orglett.8b04101 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters iPKS encoding genes other than usorB, which were not clustered with usorA, were involved in the biosythesis of 1−4. The isolated compounds were screened for their cytotoxic activities (Figure 5a). The bisorbicillinoid 1 showed
■
Experimental procedures, 1D and 2D NMR, IR, UV, and MS spectra for 1−4 (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Jungui Dai: 0000-0003-2989-9016 Ligang Zhou: 0000-0003-4681-191X Author Contributions §
These authors contributed equally.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (Nos. 31471729 and 31271996), and the National Key R&D Program of China (Nos. 2017YFD0200501 and 2017YFC1600905).
■
(1) Harned, A. M.; Volp, K. A. Nat. Prod. Rep. 2011, 28, 1790−1810. (2) Meng, J.; Wang, X.; Xu, D.; Fu, X.; Zhang, X.; Lai, D.; Zhou, L.; Zhang, G. Molecules 2016, 21, 715. (3) (a) Nicolaou, K. C.; Vassilikogiannakis, G.; Simonsen, K. B.; Baran, P. S.; Zhong, Y.-L.; Vidali, V. P.; Pitsinos, E. N.; Couladouros, E. A. J. Am. Chem. Soc. 2000, 122, 3071−3079. (b) Hong, R.; Chen, Y.; Deng, L. Angew. Chem., Int. Ed. 2005, 44, 3478−3481. (c) Nicolaou, K. C.; Simonsen, K. B.; Vassilikogiannakis, G.; Baran, P. S.; Vidali, V. P.; Pitsinos, E. N.; Couladouros, E. A. Angew. Chem., Int. Ed. 1999, 38, 3555−3559. (4) Lu, S.; Sun, W.; Meng, J.; Wang, A.; Wang, X.; Tian, J.; Fu, X.; Dai, J.; Liu, Y.; Lai, D.; Zhou, L. J. Agric. Food Chem. 2015, 63, 3501− 3508. (5) Zhang, Y.; Zhang, K.; Fang, A.; Han, Y.; Yang, J.; Xue, M.; Bao, J.; Hu, D.; Zhou, B.; Sun, X.; Li, S.; Wen, M.; Yao, N.; Ma, L.-J.; Liu, Y.; Zhang, M.; Huang, F.; Luo, C.; Zhou, L.; Li, J.; Chen, Z.; Miao, J.; Wang, S.; Lai, J.; Xu, J.-R.; Hsiang, T.; Peng, Y.-L.; Sun, W. Nat. Commun. 2014, 5, 3849. (6) 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−W243. (7) Salo, O.; Guzman-Chavez, F.; Ries, M. I.; Lankhorst, P. P.; Bovenberg, R. A. L.; Vreeken, R. J.; Driessen, A. J. M. Appl. Environ. Microbiol. 2016, 82, 3971−3978. (8) Abe, N.; Yamamoto, K.; Hirota, A. Biosci., Biotechnol., Biochem. 2000, 64, 620−622. (9) Andrade, R.; Ayer, W. A.; Mebe, P. P. Can. J. Chem. 1992, 70, 2526−2535. (10) Crawford, J. M.; Thomas, P. M.; Scheerer, J. R.; Vagstad, A. L.; Kelleher, N. L.; Townsend, C. A. Science 2008, 320, 243−246. (11) (a) Fahad, A. a.; Abood, A.; Fisch, K. M.; Osipow, A.; Davison, J.; Avramovic, M.; Butts, C. P.; Piel, J.; Simpson, T. J.; Cox, R. J. Chem. Sci. 2014, 5, 523−527. (b) Guzmán-Chávez, F.; Salo, O.; Nygård, Y.; Lankhorst, P. P.; Bovenberg, R. A. L.; Driessen, A. J. M. Microb. Biotechnol. 2017, 10, 958−968.
Figure 5. Cytotoxic activities: (a) IC50 values of 1−3; (b) compound 1 induced apoptosis in BGC823 cells (cells in the lower-right and upper-right quadrant are early- and late-apoptotic cells, respectively).
pronounced cytotoxicity against the five tested tumor cell lines, with IC50 values in the range of 4.48−18.6 μM, while the sorbyl-anthraquinones 2 and 3 displayed a weaker effect. However, the sorbyl-naphthoquinone 4, and oxosorbicillinol were inactive (IC50 > 50 μM). Compound 1 was further tested for its influence on the cellcycle progression with the gastric cancer cell line BGC823. Interestingly, compound 1 markedly induced G0/G1, and G2/ M-phase cell-cycle arrest (Figure S9 in the Supporting Information). Compound 1 was also investigated for its effect on apoptosis in BGC823 cells, as cell shrinkage and detached from culture surface was observed after treatment with 1. The apoptotic rate of BGC823 cells was examined using flow cytometry. Compared to the control group, treatment with 1 at 9 μM for 48 h induced significant apoptosis incidence in BGC823 cells (74.7%) (Figure 5b). Moreover, treatment with 1 altered the expression levels of cleaved caspase-3 and PARP (data not shown), suggesting the caspase-mediated apoptotic pathway was involved in the induced apoptosis of BGC823 cells. Further studies are required to gain a deeper understanding of the cytotoxicity. In summary, ustilobisorbicillinol A (1) had an intriguing structure and showed notable cytotoxicity. Hence, it could be an attracting candidate for further development as an antitumor agent, and also an interesting target for synthetic and biosynthetic studies.
■
REFERENCES
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b04101. D
DOI: 10.1021/acs.orglett.8b04101 Org. Lett. XXXX, XXX, XXX−XXX
