Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Peniroquesines A−C: Sesterterpenoids Possessing a 5−6−5−6−5Fused Pentacyclic Ring System from Penicillium roqueforti YJ-14 Jia-Peng Wang,† Jing Yu,† Yan Shu, Ya-Xian Shi, Ping Luo, Le Cai,* and Zhong-Tao Ding* Key Laboratory of Functional Molecules Analysis and Biotransformation, Yunnan Provincial Department of Education, School of Chemical Science and Technology, Yunnan University, Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Kunming 650091, China
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S Supporting Information *
ABSTRACT: Three novel sesterterpenes, peniroquesines A− C, possessing an 5−6−5−6−5-fused pentacyclic ring system, were isolated from the fungus Penicillium roqueforti YJ-14 by solid fermentation. Their structures were initially investigated in detail by NMR spectra and HR-ESI-MS and were further confirmed by chemical reaction experiments as well as by Xray analysis. Furthermore, a plausible biosynthetic pathway was investigated by isotope-labeling experiments. esterterpenes, which are derived from two five-carbon isoprene units, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), are a relatively rare group of pentaprenyl terpenoids that exhibit various bioactivities such as anti-inflammatory, cytotoxic, anticancer, antimicrobial, antitubercular, and antibiofilm activities as well as inhibitory activity against a variety of biological systems.1 Penicillium roqueforti, a fungus found in moldy grains and silage,2 is known to produce indole alkaloids3 and sesquiterpenoids such as PR toxin.3a,4 However, there have been few reports about sesterterpenes produced by Penicillium. Herein, we report the isolation, structure elucidation, biosynthetic pathway, and biological activity of three novel sesterterpenes (peniroquesines A−C, Figure 1) from the
S
ments. The bioactive assays of compounds 1 and 2 showed strong anti-inflammatory activities. Compound 1 was isolated as a white amorphous powder, and its molecular formula was deduced to be C25H40O2 by HRESI-MS at m/z 395.2923 (C 25 H 40 O 2 Na) (calcd for C25H40O2Na [M + Na]+, 395.2921), indicating six degrees of unsaturation. Two olefinic carbon atoms account for one degree of unsaturation, indicating the presence of five rings in 1. The IR spectrum of 1 displays an absorption band at 3444 cm−1, which corresponds to the hydroxyl groups. The presence of the hydroxyl groups was confirmed by the signals at δC 72.1 and 74.5 in the 13C NMR spectrum. The 1H, 13C, DEPT, and HSQC NMR spectra recorded in CDCl3 indicate the presence of 25 carbons (Table S1), including six methyl groups [δH 1.14 (H3-23), 1.12 (H3-22), 0.93 (H3-24), 0.91 (H3-21), 0.86 (H325), 0.77 (H3-20); δC 33.0 (C-23), 30.0 (C-24), 24.4 (C-25), 19.9 (C-22), 18.5 (C-21), 13.7 (C-20)], six methylenes [δH see Table S1; δC 44.5 (C-13), 41.8 (C-9), 41.7 (C-16), 39.9 (C17), 30.0 (C-4), 25.2 (C-5)], eight methines [δH 5.52 (H-1, olefinic), 3.62 (H-12, oxygenated), 3.39 (H-8, oxygenated), 1.76 (H-3), 1.46 (H-6), 2.35 (H-10), 1.60 (H-14), 1.22 (H18); δC 133.8 (C-1, olefinic), 74.5(C-12), 72.1 (C-8), 57.4 (C18), 56.2 (C-6), 53.2 (C-14), 51.4 (C-10), 38.9 (C-3)], and five quaternary carbons [δC 147.1 (C-2, olefinic), 49.2 (C-11), 47.2 (C-7), 43.5 (C-19), 42.6 (C-15)]. These results strongly suggest that 1 was a pentacyclic sesterterpene. The planar structure of 1 was constructed by 2D NMR analyses in which three spin systems a−c (Figure 2) were readily identified by analysis of the 1H−1H COSY correlations (Figure S7) of H-12/H2-13 (δH 1.53−1.57, 2H), H2-16 (δH 1.53, 1.65)/H2-17 (δH 1.43, 2H), and H-14/H-10/H2-9 (δH 0.95, 2.17)/H-8/H-6/H2-5 (δH 1.71, 1.90)/H2-4 (δH 1.37,
Figure 1. Structures of peniroquesines A−C (1−3).
fungus P. roqueforti YJ-14 by solid-state fermentation (SSF), which is an efficient fermentation method with a series of advantages such as producing structurally complex metabolites because of its longer metabolic lifetime.5 The fungus P. roqueforti YJ-14 was separated from A. vilmorinianum Kom. Peniroquesines A−C were characterized as sesterterpenes possessing an 5−6−5−6−5-fused pentacyclic ring system based on NMR spectroscopy, MS, Mosher’s method, and single-crystal X-ray crystallography. Furthermore, a mechanistic study of its cyclization from geranylfarnesyl pyrophosphate (GFPP) was conducted using isotope-labeling experi© XXXX American Chemical Society
Received: August 7, 2018
A
DOI: 10.1021/acs.orglett.8b02534 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 2. Key 1H−1H COSY (blue line), HMBC (red arrows), and NOESY (pink arrows) correlations of peniroquesine A (1).
Figure 3. X-ray crystal structure of peniroquesine C (3).
1.93)/H-3/H3-20, respectively. The presence of a 1,1dimethylcyclopentane (ring A) was deduced from the HMBC correlations (Figures 2 and S8) of H3-23/C-16, C15, C-18, and C-13; H3-24/C-17, C-19, C-18, and C-25; H325/C-17, C-19, C-18, and C-24; and H2-16 and H2-17/C-15, C-18 and C-19. Then the direct connection between C-18 (methine) in ring A and C-14 (tertiary carbon) in ring B was confirmed by the HMBC correlations of H-14/C-18, C-19, and C-15. These correlations combined with the HMBC correlations of H3-22/C-1, C-12, C-11, and C-14; H2-13/C-15, C-16, C-18, C-23, C-12, and C-11; H-12/C-13, C-15, C-11, and C-14; H-18/C-11, C-15, C-14, and C-13; and H-14/C-11 and C-12 indicated that 1 possesses a six-membered B ring. Furthermore, the chemical shift of C-12 is δC 74.5, indicating that a hydroxyl group was located at C-12. The HMBC correlations of H-1/C-2, C-10, C-11, and C-14; H-10/C-1 and C-2; H-14/C-11; H3-22/C-1, C-11, and C-14; H-10/C-1, C-2, and C-14 established a cyclopentene ring C. The HMBC correlations of H3-21/C-2, C-7, and C-6; H-8/C-6 and C-7; H2-9/C-10, C-2, C-8, and C-6 confirmed a cyclohexane ring D. The presence of a hydroxyl substituent at C-8 was identified based on the 13C chemical shift δC 72.1. The methylcyclopentane ring E was deduced from the HMBC corrections of H3-21/C-2, C-3, C-6, and C-7; H3-20/C-3, C-7, and C-4; H-3/ C-7 and C-6; H2-4/C-7 and C-3; and H2-5/C-3, C-7, and C-6. Accordingly, the planar structure of compound 1 was identified as a 5−6−5−6−5-membered pentacyclic sesterterpene (rings A−E). The relative configuration of 1 was deduced on the basis of the NOESY correlations (Figures 2 and S10) of H3-23/H-12 and H-18; H-18/H3-24 and H-10; H-10/H-8; and H-8/H-3, which indicated that these groups are α oriented, while the correlations of H3-25/H-14; H-14/H3-22; H3-22/H-1; H-1/ H3-21; and H3-21/H3-20 and H-6 revealed that these groups are β oriented. Therefore, the relative configuration of 1 was determined to be 3S*,6S*,7R*,8S*,10S*,11S*,12R*,14R*,15S*,18S*. The absolute configuration of 1 (3S,6S,7R,8S,10S,11S,12R,14R,15S,18S), named peniroquesine A, was determined on the basis of Mosher’s method6 (see the Supporting Information), analysis of 2 using Mosher’s method, the single-crystal X-ray structure of 3 (Figure 3), and the fact that these compounds are all produced by the fungus P. roqueforti YJ-14 and share the same fused pentacyclic ring system and relative configurations at C-3, C-6, C-7, C-10, C11, C-12, C-14, C-15, and C-18, which were determined by 1D and 2D NMR experiments. The molecular formula of compound 2 was established based on its HRESIMS [M + Na]+ ion at m/z 379.2971 to be C25H40O (calcd for C25H40ONa [M + Na]+, 379.2971), indicating this compound contained one fewer hydroxyl group than 1. The 1H and 13C NMR spectra (Table S1) in CDCl3
suggested that 2 had the same carbon skeleton as 1. The chemical shift of C-8 (δC 29.0) in 2 and the absence of oxygenated proton H-8 and oxygenated carbon C-8 in 1 suggested that oxygenated methine CH-8 in 1 was replaced with a methylene, which was further confirmed by the 1H−1H COSY (Figures S2 and S14) correlations of H-10 (δH 2.23)/ H2-9 (δH 0.86, 1.91)/H2-8 (δH 1.26, 1.93)/H-6 (δH 1.64). The relative stereochemistry of 2 was deduced on the basis of its NOESY spectroscopic data (Figure S3 and S17): H3-23 (δH 1.15)/H-12 (δH 3.62) and H-18 (δH 1.23), H-18/H3-24 (δH 0.91) and H-10 (δH 2.23), and H-10/H-12 and H-3 (δH 1.96), which indicated that these groups are α oriented, while the correlations of H3-25 (δH 0.84)/H-14 (δH 1.51), H-14/H3-22 (δH 1.10), H3-22/H-1 (δH 5.47), H-1/H3-21 (δH 0.85), and H3-21/H3-20 (δH 0.77) and H-6 (δH 1.64) revealed that they are β oriented. Therefore, the relative configuration of 2 was determined to be 3S*,6R*,7R*,10S*,11S*,12R*,14R*,15S*,18S*. Mosher’s method6 (see Supporting Information) allowed the absolute configuration of 2, named peniroquesine B, to be assigned as 3S,6R,7R,10S,11S,12R,14R,15S,18S (Figure S4). Compound 3 has a molecular formula of C25H40O2, which was confirmed by the HRESIMS ion at m/z [M + Na]+ 395.2923 (calcd for C25H40O2Na [M + Na]+, 395.2921). Comparing the 1H and 13C NMR data of 3 (Table S1) with those of 2, the absence of a methyl CH3-23 in 2 and the presence of the oxygenated methylene CH2-23 (δH 3.71, 3.95; δC 69.6) in 3 indicated that a hydroxyl group was located at methyl C-23 of 3, which was further supported by the HMBC correlations (Figure S22) of H2-23/C-13 (δC 40.0), C-15 (δC 48.9), C-16 (δC 37.2), and C-18 (δC 53.0). The relative configuration was deduced on the basis of the following NOE correlations (Figure S24): H2-23/H-12 (δH 4.14) and H-18(δH 1.47), H-18/H-10 (δH 2.41) and H3-24 (δH 1.03), and H-10/ H-12 and H-3 (δH 1.89), which indicated that these groups are α oriented, while the correlations of H3-25 (δH 1.04)/H-14 (δH 1.73), H-14/H3-22 (δH 1.53), H3-22/H-1 (δH 5.91), H-1/ H3-21 (δH 0.91), H3-21/H3-20 (δH 0.71), and H-6 (δH 1.65) revealed that these groups are β oriented. Thus, the relative configuration of 3 was determined to be 3S*,6R*,7R*,10S *,11S*,12R*,14R*,15R*,18S*. Fortunately, crystals suitable for X-ray diffraction were obtained by slow evaporation from a chloroform solution over 3 days. Therefore, the absolute configuration of 3 was determined7 (see the Supporting Information) to be 3S,6R,7R,10S,11S,12R,14R,15R,18S, and 3 was named peniroquesine C. To investigate the assembly of the carbon skeleton during cyclooctatin biosynthesis, we uniformly administered either [1-13C]-sodium acetate and [U-13C6]-glucose to potato cultures of peniroquesine-producing P. roqueforti YJ-14. The resulting 13C enrichments and the 13C−13C coupling patterns were determined by a combination of one-dimensional 13C B
DOI: 10.1021/acs.orglett.8b02534 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Scheme 1. (A) Summary of the Isotope Tracing Experiments with [U−13C6]-Glucose for the Biosynthesis of 1. (B) Proposed Cyclization Mechanism for the Generation of Peniroquesines
Table 1. IC50 Values (μM) for Cytotoxic Activity and Inhibitory Effects on LPS-Induced NO Production in RAW 264.7 Macrophages of 1−3 IC50 (μM) compd 1 2 3 DDPa Taxola L-NMMAb
HL-60 30.73 16.52 11.99 3.52
