Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
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Talaromyolides A−D and Talaromytin: Polycyclic Meroterpenoids from the Fungus Talaromyces sp. CX11 Xun Cao,† Yutong Shi,† Xiaodan Wu,† Kuiwu Wang,§ Shaohua Huang,∥ Hongxiang Sun,⊥ Jeroen S. Dickschat,‡ and Bin Wu*,† †
Ocean College, Zhejiang University, Hangzhou 310058, China Kekulé-Institute for Organic Chemistry and Biochemistry, University of Bonn, 53121 Bonn, Germany § Department of Applied Chemistry, Zhejiang Gongshang University, Hangzhou 310058, China ∥ Institute of Drug Discovery Technology, Ningbo University, Ningbo 315000, China ⊥ College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
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ABSTRACT: Talaromyolides A−D (1−4) and talaromytin (5) were isolated from a marine fungus Talaromyces sp. CX11. Their structures were unambiguously determined by nuclear magnetic resonance (NMR), mass spectrometry, X-ray crystallography experiments, and time-dependent density functional theory electronic circular dichroism calculations. Talaromyolides A and D represent two novel carbon skeletons. Talaromytin exhibits two slowly interconverting conformers in DMSO-d6 and CH3OH-d4 that were studied by temperature-dependent NMR experiments. Talaromyolide D exhibits potent antiviral activity against pseudorabies virus (PRV) with a CC50 value of 3.35 μM.
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eroterpenoids are hybrid natural products partially derived from the polyketide and partially from the terpenoid pathway.1,2 Recently, meroterpenoids have attracted attention because of their chemical diversity and biological activities.3,4 The genus Talaromyces is known to produce meroterpenoids, polyketides, and furanosteroids with novel chemical structures.5,6 The meroterpenoid class isolated from this genus usually possesses congested polycyclic skeletons, such as berkeleyacetals with 6/7/6/5/6 cyclic system6 and 6/6/6/6 cyclic talarolutins.7 Over the past few decades, only 15 meroterpenoids constructed with drimane-type sesquiterpene moieties and C10 polyketide units have been reported.8−11 In this study, a chemical investigation on the strain CX11 resulted in the isolation of five new meroterpenoids. Here, we report on the isolation, structure elucidation, a biosynthetic rationale, and the biological activities of these compounds. Talaromyolide A (1, Figure 1) was obtained as a yellow oil and possessed the molecular formula C23H28O8 as determined by HRESIMS (m/z 433.1857 [M + H]+, calc. for C23H29O8+: m/ z 433.1857), indicating ten degrees of unsaturation in 1. The 13C NMR spectrum showed the presence of 10 signals for isocoumarin substructure usually derived from the precursor 6-hydroxymellein, which is a structural element also observed in other meroterpenoids from this fungus such as the chrodrimanins8−10 and verruculide A.11 The remaining 13 resonances corresponded to a bisnor-sesquiterpene moiety. The presence of a hemiketal or ketal functional groups was supported by the chemical shifts of C-20 (δC 99.5) and C-25 (δC 99.7). The carbonyl group and the benzene ring in the isocoumarin unit accounted for five degrees of unsaturation, thus requiring the presence of five additional rings in 1. © XXXX American Chemical Society
Figure 1. Structures of meroterpenoids 1−5 from Talaromyces sp.
The planar structure of 1 was further elucidated by 2D-NMR spectroscopic analysis. The 1H,1H-COSY displayed cross-peaks for H-9/H-10 and H-9/H3-12 that together with HMBC correlations from H-9/H-10 to C-2 confirmed the presence of the isochromanone moiety (Figure S1). HMBC correlations from H2-15/H2-21/H2-26 to C-16, from H2-15/H2-21/H3-28 to C-19, and from H2-21/H3-28 to C-20 established the 6/6/6/5/ 5/5 polycyclic skeleton with four heterocycles, bridging linkages between C-16 and C-25 and between C-20 and C-25 with Received: July 16, 2019
A
DOI: 10.1021/acs.orglett.9b02466 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters oxygen atoms, respectively. The 1H,1H−COSY cross-peaks for H-30/H 3 -31 and H-30/H 3 -32 along with the HMBC correlations from H-30/H3-31/H3-32 to C-25 demonstrated the attachment of an isopropyl group at C-25, resulting in the completed constitutional formula of 1. The relative configuration of 1 was determined by ROESY (Figure S6). The diagnostic 1,3-diaxial correlations of H3-23/ H2-15β and H3-28/H2-15β and the correlations of H3-23/H221β and H3-28/H2-21β placed these hydrogens and methyl groups in one hemisphere, assigning Me-23 and Me-28 in axial position, while the correlation of H-10/H2-15α assigned the H10 as α-oriented. The absolute configuration of 1 was determined from the experimental electronic circular dichroism (ECD) spectrum in comparison to Boltzmann-weighted timedependent density functional theory (TDDFT) calculated spectra (Figure 2), confirming the absolute configuration of (9R,10R,16S,17R,19R,20S,25R)-1.
Figure 3. Single-crystal X-ray structure of 2.
Talaromyolide C (3, Figure 1) was isolated as a colorless powder and assigned a molecular formula of C25H32O7 based on the HRESIMS spectrum (m/z 445.2222 [M + H]+, calc. for C25H33O7+: m/z 445.2221). The 1H- and 13C NMR data of 3 closely resembled those of 2, revealing that both share the same carbon skeleton. A detailed comparison disclosed the absence of the hydroxy group at C-10. The structure of 3 was further supported by key 1 H,1H−COSY, HMBC, and ROESY correlations (Figures S3 and S9). The absolute configuration of 3 was established by TDDFT-calculation of ECD spectra and comparison to the experimental ECD curve (Figure S13), resulting in the structure of (9R,16S,17R,19R,20S,25R)-3. Compounds 2 and 3 represent the first examples of meroterpenoids with a seco-drimane sesquiterpene moiety and a C10 polyketide in their structures. Talaromyolide D (4, Figure 1) possessed a molecular formula of C25H34O5 on the basis of HRESIMS data (m/z 415.2481 [M + H]+, calc. for C25H35O5: m/z 415.2479), indicating nine double bond equivalents. A detailed interpretation of the 1Dand 2D-NMR spectra and comparison to 2 suggested that compound 4 also possessed an isochromanone system connected to two more six-membered rings (Figure S4). HMBC correlations from H-24 to C-18/C-23/C-25/C-28 and from H-27 to C-28/C-29 together with the 1H,1H-COSY system of H-24/H2-26/H-27 established an unprecedented dimethylcyclobutanol subunit. A ROESY experiment revealed the partial relative configuration of 4, with cross-peaks for H3-22/H2-14β, H3-23/H2-14β, H-15/H2-14α, H-15/H-24, and H-24/H-27 (Figure S9). The (S)- and (R)-MTPA esters of 4 were prepared, resulting in ΔδH values (ΔδH = δS − δR) as indicated in Figure S11. Referring to the rule of modified Mosher’s method,12,13 the absolute configuration of C-27 was deduced to be R. The Boltzmann-averaged calculated ECD spectra of 4 displayed a highly matching curve to the experimental spectrum for (9R,15R,16R,18R,24R,27R)-4 (Figure S14). Talaromytin (5, Figure 1) was obtained as colorless crystals. Its molecular formula was deduced to be C30H32O13 on the basis of HRESIMS data (m/z 623.1738 [M + Na]+, calc. for C30H32O13Na+: m/z 623.1735), implying 15 degrees of unsaturation. The 1H NMR spectrum of 5 at room temperature (Figure 4) suggested the presence of two isomers in a ratio of 3:2, but various attempts to separate these compounds failed. Conclusively, we supposed that talaromytin was a pair of interconverting conformers, a phenomenon that is sometimes observed for terpenoid natural products14−17 A series of temperature-dependent 600 MHz 1H NMR spectra confirmed this hypothesis. Increasing the temperature to 323 K resulted in peak broadening, while at 343 K coalescence was observed. Further increases to 373 and 393 K gave sharp peaks (fast exchange regime).
Figure 2. Experimental ECD spectrum and calculated ECD spectrum of (9R,10R,16S,17R,19R,20S,25R)-1.
Talaromyolide B (2, Figure 1) was obtained as colorless crystals with the molecular formula C25H32O8, as inferred from its HRESIMS (m/z 461.2171 [M + H]+, calc. for C25H33O8+: m/ z 461.2170), corresponding to ten sites of unsaturation. The 1DNMR spectroscopic data of 2 were very similar to those of 1, except for the presence of two additional methylenes that were also observable in the 13C NMR spectrum (Figure S23). This analysis indicated that 2 was a structurally related meroterpenoid with a complete seco-drimane unit and an isocoumarin substructure, which was confirmed by 1H,1H-COSY correlations between H2-21/H2-22 and H2-26/H2-27 along with HMBC signals from H2-15/H2-22/H2-27 to C-16 and H2-21/H2-26/ H2-27 to C-19, allowing to place the additional methylene groups as compared to 1 (Figure S2). Elucidation of the full planar structure of 2 resulted in a fused 6/6/6/6/6/6 hexacyclic skeleton with four heterocyclic rings. ROESY experiments revealed the relative configuration of 2, demonstrating that H3-23, H3-28, H2-15β, and H2-21β are located in the same hemisphere, while H2-21α, H2-15α, and H10 are on the opposite site of the molecule (Figure S7). The absolute configuration was tentatively determined from its single-crystal X-ray structure (Cu Kα) as (9R,10R,16S,17R,19R,20S,25R)-2 (Figure 3), based on a poor Flack parameter of 0.2(4), but was confirmed by a comparison of the experimental ECD spectrum with the TDDFT calculated ECD spectra (Figure S12). B
DOI: 10.1021/acs.orglett.9b02466 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
Figure 6. Two conformers of 5 with different orientations of the ester side chain (structures generated with the MM2 function of ChemBio3D Ultra 13.0).
Scheme 1. Plausible Biosynthetic Pathway of Talaromyolides B−D (2−4)a Figure 4. Variable temperature 1H NMR spectra of 5 (in DMSO-d6).
Analysis of the 13C NMR data showed that 5 possessed five carbonyl carbons and six olefinic carbons, suggesting a heptacyclic nature in 5. The 1H NMR data displayed one aldehyde proton, five olefinic protons including one exocyclic methylene group, four oxymethines, one oxymethylene, and six methyl groups. Overall, the 1H NMR spectrum displayed a similar feature to that of the known compound penicianstinoid A.1,18 The obvious dissimilarities were the lack of two oxygen bridges between C-8/C-33 and C-7/C-31, the presence of one aldehyde and a hydroxyl group at C-14. The 1H,1H-COSY correlation of H-7/H-11 and the HMBC correlations from H226 to C-8, H-33 to C-29, and H3-41 to C-31 unambiguously confirmed the planar structure of 5. Despite the existence of the two conformers observed at room temperature, after repeated attempts we obtained single crystals of 5 that were subjected to an X-ray diffraction experiment with Cu Kα radiation (Figure 5). The absolute configuration
Figure 5. Single-crystal X-ray structure of 5. a
therefore was determined as (7R,8S,9S,11S,14R,15R,31R,33S,37R)-5. Since the polycyclic core structure of 5 is likely conformationally rigid, the two observed conformers may likely be represented by different orientations of the ester side chain (Figure 6). A plausible biosynthetic hypothesis for compounds 1−4 is presented in Schemes 1 and S1. The pathway starts at 6hydroxymellein (6) by electrophilic aromatic substitution with farnesyl diphosphate (FPP) by a prenyltransferase (PT) to yield 7. The protonation-induced terpene cyclization to 8 by a class II terpene cyclase (TC) may be followed by a cytochrome P450
For talaromyolide A, see Scheme S1.
(CYP450) mediated epoxidation to 9. The epoxide ring opening leads to the cationic intermediate A+ that can stabilize by deprotonation with formation of a cyclobutane ring to give 4 (path b) or by a 1,2-hydride shift to yield 10 (path a, epoxideketone rearrangement). Further CYP450 oxidation to 11 and likely spontaneous ketal formation leads to 3 that can be further oxidized to 2. The biosynthesis of 1 that is obviously biosynthetically connected to this pathway requires the loss of two carbons derived from FPP by an unclear mechanism. The starting point toward 1 may also be cation A+ that can undergo C
DOI: 10.1021/acs.orglett.9b02466 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
Chemistry and Ocean College, Zhejiang University) for their kind help on the measurement of NMR experiments.
deprotonation with cyclopropane ring formation to 12 (path c). A mechanistic hypothesis for the loss of two carbons involving oxidative chemistry and spontaneous decarboxylations is shown in Scheme S1. All five compounds were evaluated for their biological activities in cytotoxicity assays, demonstrating that all compounds were inactive against the HL-60, K562, MGC803, BEL-7402, SH-SY5Y, HCT-116, MDA-MB-231, A549, MCF-7/ADM, HO8910, U87, and NCI-H1975 cell lines. Only compound 4 exhibited an antiviral activity against pseudorabies virus (PRV) with a CC50 value of 3.35 μM. In summary, talaromyolides A−D (1−4) and talaromytin (5), five novel meroterpenoids, were obtained from the fungus Talaromyces sp. CX11. Their absolute configurations were determined by X-ray anomalous dispersion, comparison of calculated and experimental ECD spectra, or Mosher ester analysis. Although none of these methods is absolutely reliable, for all compounds 1−4, consistent absolute configurations were found, and the absolute configuration of 5 is in good agreement with those reported for other members of the compound family. Notably, compound 1 must have lost two carbons from the FPP chain. This finding will be interesting to study in depth, but will require access to the biosynthetic gene cluster and the encoded enzymes of the pathway. Also the formation of compound 4 seems to be unusual, although it is easier to understand. Its unique structural element of the dimethylcyclobutanol subunit may be important for its bioactivity against PRV and makes it a valuable target for future chemical synthesis and biosynthetic studies.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b02466. General information on the experiment, experimental procedures, characterization data, biological activity assays, and NMR spectra for all the new compounds (PDF) Accession Codes
CCDC 1939890−1939891 contain 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
[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 Author
*E-mail:
[email protected]. ORCID
Jeroen S. Dickschat: 0000-0002-0102-0631 Bin Wu: 0000-0002-7638-2696 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Key R&D Program of China (No. 2018YFC0311002) and NSFC (Nos. 81573306 and 41876148). We thank Yaqin Liu and Ying Liu (Department of D
DOI: 10.1021/acs.orglett.9b02466 Org. Lett. XXXX, XXX, XXX−XXX
