Letter Cite This: Org. Lett. 2018, 20, 1806−1809
pubs.acs.org/OrgLett
Highly Photosensitive Poly-Sulfur-Bridged Chetomin Analogues from Chaetomium cochliodes Meng-Hua Wang,† You-Cai Hu,‡ Bing-Da Sun,§ Meng Yu,† Shu-Bin Niu,∥ Zhe Guo,† Xiao-Yan Zhang,† Tao Zhang,† Gang Ding,*,† and Zhong-Mei Zou*,† †
Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, P.R. China ‡ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, P.R. China § Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, P.R. China ∥ Department of Pharmacy, Beijing City University, Beijing 100083, P.R. China S Supporting Information *
ABSTRACT: The highly photosensitive characteristic of poly-sulfide chetomins was first unveiled, and four new unstable analogues, chetomins A−D (1−4), with significant cytotoxicity were successfully purified in darkness. The visiblelight-induced desulfurization and intermolecular disproportionation were revealed to initiate the interconversion of chetomin analogues, which explained the long-recognized puzzle of rarity and instability of chetomin analogues.
C
hetomin (5) was first isolated in 1944 by Waksman and Bugie from the fungus Chaetomium cochliodes with a strong cytotoxicity by acting on the transcription factor, hypoxiainducible factor 1 (HIF-1).1 This member of mycotoxins possesses an asymmetrically dimeric epidithiodiketopiperazine (ETP) skeleton linked by a unique N−C bond, which is different from those symmetric dimers connected by a C−C bond (Figure 1). Due to its complex structural features, together with congested stereogenic centers and highly acid-, base-, and
redox-sensitive functional groups, 74 years had passed without a total synthesis report on chetomin, though two groups totally synthesized (+)-11,11′-dideoxyverticillin A and chaetocin (Figure S1).2 Williams et al. recently reported the enantiospecific synthesis of desthiochetomin, whereas the introduction of two sulfur bridges to the ETP core of chetomin remained unsolved.3 Different poly-sulfur-bridged ETP dimers were found in structures of verticillins, chaetocins, and leptosins,4 whereas until now, only two chetomin analogues were reported: chetomin (5, S2−S2, the only stable one of chetomin family) and chetoseminudin A (S2−S3) .5 Though Fujimoto et al. reported the isolation of chetoseminudin A, they did not provide the original NMR spectra of pure chetoseminudin A.5 Wu et al. used the HPLC-ESI-MS/MS method to detect the existence of several poly-sulfur-bridged chetomin analogues in the crude extract of C. cochliodes.6 Li et al. observed a S4−S4 bridged analogue in the extract of C. cochliodes, which was easily transformed into other derivatives.7 However, the underlying mechanism behind this phenomenon failed to be further investigated, and no pure poly-sulfur chetomin analogues were purified. These results implied that chetomin analogues must possess different physicochemical properties, compared with other sulfur-bridged natural or synthesized compounds (Figure S1),2−4,8 which led to their instability. Those interesting and unusual phenomena of chetomins attracted us to solve this problem.
Figure 1. Structures of different dimeric ETPs.
Received: January 28, 2018 Published: March 14, 2018
© 2018 American Chemical Society
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DOI: 10.1021/acs.orglett.8b00304 Org. Lett. 2018, 20, 1806−1809
Letter
Organic Letters
istics (Figure S13) and 2D NMR data of 1. Thus, the planar structure of 1 was determined (Figure 3).
To gain access to poly-sulfide chetomin analogues, the UPLCDAD-Q-TOF-MS/MS technique was initiated to analyze the crude extract of fungus C. cochliodes, and a series of secondary metabolites with unique molecular weights (parent ions: 861, 829, 797, 765, 733) were detected (Figure S2). The fragment ions displayed the diagnostic characteristics of neutral loss of sulfur atoms (64/SS, 66/HSSH). Detailed analysis of the MS rule revealed that these natural products might be poly-sulfur-bridged chetomin analogues (Supporting Information (SI)). However, except for chetomin (m/z 733 [M + Na]+), the NMR spectrum of other “so-called pure compounds” collected from a single HPLC peak always gave mixed signals (Figure S3). Then, HPLC and UPLC-MS analyses revealed that the “so-called pure compound” had transformed into several analogues for some unknown reason (Figures S4 and S5). Obviously, the rapid transformation interpreted the failure to purify and synthesize poly-sulfide chetomin analogues. Oxygen, temperature, and light were postulated to be factors leading to the instability of chetomin analogues (Figure 2). First,
Figure 3. 1H−1H COSY, key HMBC, and ROESY correlations of 1.
The relative configuration of 1 was characterized by ROESY data, which confirmed the same correlations as those of chetomin 5. Yet, the absolute configurations could not be determined by NMR methods. 1 was converted to its corresponding dethiotetra(methylthio) product (7) by treatment with NaBH4 and MeI in pyridine with retention of the same stereochemistry (Scheme S1).9 The NMR data and optical rotation degree of the reaction product (7) were identical to those of compound 6, demonstrating the same structure possessed by 6 and 7. Compound 6 was also isolated from the same fungus, and its absolute configuration was determined as 3S,5R,10bS,11aS,3′S,6′S by X-ray crystallography (Figure 4), which established the stereochemistries of 1.
Figure 2. Investigation of different factors causing instability.
N2 protection was adopted during the whole purification process, but the collected “pure compound” from semipreparative HPLC was still a mixture. Then, the purified samples were conserved in low temperature (ice bath), whereas the samples still deteriorated. At last, compound purification proceeded in darkness. Strikingly, NMR spectra and HPLC analysis confirmed the purity of those compounds. Thus, light was demonstrated to be the key inducer leading to the instability and interconversion of poly-sulfide-bridged chetomin analogues. Further measurement of photosensitivity showed that even weak ambient light could trigger the interconversion in a very short time. After the problem of the unstable characteristic was solved, four new unstable poly-sulfide analogues named chetomins A−D (1−4) together with the stable compounds chetomin (5) and dethiotetra(methylthio)chetomin (6) were successfully purified and analyzed by 1D and 2D NMR in darkness. Compound 1 was isolated as a white amorphous powder. The molecular formula C31H30N6O6S6 was established by HR-ESIMS (m/z 797.0447 [M + Na]+, calcd 797.0449). The NMR spectra of 1 exhibited two sets of indole systems, two diketopiperazine cores, three N-methyls, and four methylene groups, indicating 1 to be a chetomin-type analogue. The NMR data of 1 showed resemblance to those of chetomin 5, except the NMR signals of C-3′, C-4′, C-6′, C-7′, C-8′, C-9′, CH2OH-3′, CH3-2′, and CH3-5′ were shifted significantly (Table S5). These NMR and chemical formula differences along with the fragmentation pattern suggested the disulfide bridge (-S2-) of subunit A in chetomin might be replaced with a tetrasulfide bridge (-S4-) in 1. This hypothesis was further supported by detailed analysis of the mass spectrometric cleavage character-
Figure 4. X-ray structure of 6 (ellipsoids are set at 50% probability level).
Compounds 2−4 were characterized similarly by HR-MS and NMR experiments (SI). The methylation reaction of other unstable analogues (2−4) was also performed in the same way as that of 1, and all of them resulted in the same product. Thus, this series of poly-sulfide secondary metabolites shared the same stereochemistry as their methylated homologue 6. With four pure chetomin analogues in hand, the interconversion mechanism was investigated. Meltzer et al. found that the dissociation of organic disulfides could be promoted by lamp light, and the scission of the S−S bond in some cyclic disulfide induced by irradiation was also revealed.10 Compound 3 possessing S4−S4 bridges quickly transformed into S4−S3 and S4−S2 analogues mainly by loss of one or two sulfur atoms when exposed to light,11 whereas compound 1 (S4−S2) produced S2− S2, S2−S3, S3−S2, S3−S3, S4−S3, and S4−S4 analogues in light 1807
DOI: 10.1021/acs.orglett.8b00304 Org. Lett. 2018, 20, 1806−1809
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Organic Letters
Figure 5. (A,B) Irradiation experiment of compound 1 with/without TEMPO analyzed by UPLC-MS (LC traces λ = 210 nm). (C) Captured molecular ion peak of the disproportionation intermediate during the irradiation process. (D) Proposed thiyl radical reaction pathway of compound 1; desulfurization (path A) and disproportionation (path B). The structures were simplified, and the irradiation-induced radicals are highlighted in green. (E) EPR spectra of compound 1 under different illumination conditions.
(Figure S18). Except for the desulfurized products of S3−S2 and S2−S2 analogues, the observation of other poly-sulfur-bridged analogues implied that different reactions, not the loss of sulfur atoms only, must happen. The trisulfide gliotoxin E turned into its disulfide and tetrasulfide analogues when dissolved in ethanol and refluxed for 2 days by intermolecular disproportionation.12 A similar reaction shaped disulfide and tetrasulfide monoacetates from trisulfide emethallicin D monoacetate in a basic hydrolytic condition (Scheme S2).13 Tobolsky et al. investigated the thermal decomposition and recombination of poly-sulfide and concluded the disproportionation of dimethyl tetrasulfide proceeded via free radicals.14 Based on the above findings, light was speculated to induce the production of free radicals which formed different chetomin analogues from a pure unstable analogue by desulfurization and intermolecular disproportionation reactions. Analysis of free radicals was an intractable issue because of their high reactivities and transient lives. EPR spectroscopy was usually a powerful tool to detect free radicals with high sensitivity.15 Herein, the pure chetomin analogues were analyzed by EPR combined with the spin-trapping agent DMPO in aqueous/acetonitrile solution. As shown in Figure 5E, the EPR spectrum of chetomin A (1, S4−S2) displayed complex signals under general illumination, whereas no signal was observed in darkness, and EPR signals attenuated rapidly after shutting off the lamp. The dynamic analysis of chetomin A (1, S4−S2) under irradiation (an ordinary flashlight as the light source) was performed with UPLC-Q-TOF-MS. The pure compound chetomin A in darkness (Figure 5A,i) began to produce trace amounts of S3−S2 and S4−S3 analogues when exposed to light for 5 s (Figure 5A,ii). Other analogues including S2−S2, S2−S3, S3− S3, and S4−S4 were also accumulated gradually with the increase of illumination time until reaching an equilibrium state (Figure
5A,v). In contrast, the light-induced interconversion was remarkedly inhibited by addition of excess TEMPO, a free radical trapper. Only a thimbleful of S3−S2 and S4−S3 analogues was generated even after 1 min continuous illumination (Figure 5B), further confirming the production of free radicals in light.14−16 Therefore, the mechanism of the unstable nature of chetomin A (1, S4−S2) was unveiled. First, homolytic cleavage of the R1−S−S−S−S−R2 triggered by light produced thiyl radicals including R1−S−S• and •S−S−R2, then the free radicals were further dissociated to generate R1−S• and R2−S•. The following spontaneous intramolecular polymerization of radicals resulted in desulfurization products chetomin (5, S2−S2) and chetomin D (4, S3−S2) (Figure 5D, path A). Second, intermolecular disproportionation of chetomin analogues also occurred. Path B shows one of the free radical reactions, which explained how chetomin A (1, S4−S2) formed chetomin C (3, S4−S4) in light. Fortunately, a weak signal of molecular ion peak (m/z 1549 [M + H]+, 1571 [M + Na]+) of the key intermolecular disproportionation intermediate was captured (Figure 5C), further supported the proposed free radical pathways. Other photoinduced disproportionation is provided in Schemes S3−S7. Moreover, the monosulfide chetomin analogues (-S-) were not detected neither in the free radical reactions nor in the extract. This might be arising from the higher dissociation energy of the C−S bond compared to that of the S−S bond,17 which also explained the stable characteristic of chetomin in the presence of light (Scheme S6), compared with other unstable analogues. Chetomin was regarded as a potential cancer chemotherapeutic agent.1 Cytotoxicity against three cancer cell lines, HepG2, MCF-7, and HeLa, was evaluated with the pure compounds 5 and 6, and with three mixed samples of those unstable ones (see SI for details). All tested samples, except for 6, displayed significant inhibitory activities at nanomolar concentrations compared with the positive control cis-platinum (Table 1808
DOI: 10.1021/acs.orglett.8b00304 Org. Lett. 2018, 20, 1806−1809
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S7). Additionally, the sulfur bridges acted as the key biological groups, and analogues possessing poly-sulfur bridges were more active than disulfide-bridged chetomin. The isolation and identification of labile natural products are always thorny issues with very limited successful results. Though naturally occurring compounds, including suffrutines A and B, malbranpyrroles A−F, and avermectins, are found to be sensitive to UV irradiation,18 this is the first report of highly visible-lightsensitive poly-sulfur-bridged natural products. The interconversion phenomenon induced by free radicals under illumination is also unraveled, which explains the long-recognized puzzle of rarity of chetomin analogues and provides an effective method to purify this unique member of heterodimeric ETPs. In addition, this report can also pave the way for the total synthesis and further biosynthetic investigation of chetomin analogues.
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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.orglett.8b00304. General experimental procedures, research of the instability, MTT assay, NMR and MS spectra for compounds 1−6 (PDF) Accession Codes
CCDC 1545668 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
You-Cai Hu: 0000-0002-3752-7485 Gang Ding: 0000-0002-8178-4788 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Professor Ren-Xiang Tan, Nanjing University, for carefully reading our manuscript and giving useful suggestions. We acknowledge financial support from The National Key Research and Development Program of China “Research and Development of Comprehensive Technologies on Chemical Fertilizer and Pesticide Reduction and Synergism” (2017YFD0201402 to D.G.), CAMS Initiative for Innovative Medicine (2017-I2M-4-004 to D.G. and H.Y.C.), the National Natural Science Foundation of China (31570340 to D.G.), the Open Funding Project of the State Key Laboratory of Mycology (No. SKLMKF201407 to S.B.D.), Beijing Nature Science Foundation (7174284 to N.S.B.), and the Chinese National S&T Special Project on Major New Drug Innovation (2017ZX09031059 to Z.T. and Z.Z.M.).
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REFERENCES
(1) (a) Waksman, S. A.; Bugie, E. J. Bacteriol. 1944, 48, 527. (b) Viziteu, E.; Grandmougin, C.; Goldschmidt, H.; Seckinger, A.; Hose, D.; Klein, 1809
DOI: 10.1021/acs.orglett.8b00304 Org. Lett. 2018, 20, 1806−1809
