English

• 1.   Introduction

  Fungi are recognized as a rich resource for structurally and biologically diverse active natural products [1, 2, 3]. Polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid metabolites are a large group of fungal metabolites that display a wide rangeof biological properties such as antitumor, antibiotic, and insecticidalactivities [4, 5, 6, 7, 8].The endophytic fungus Periconia sp. F-31 was isolated from the medicinal plant Annona muricata in Hainan Province, China. Bioassay results revealed that the extract of this strain displayed cytotoxic, antiviral, and anti-inflammatory activities. Previous chemical study of endophytic fungus Periconia sp. F-31 have led to several bioactive metabolites with diverse carbon skeletons [9, 10, 11]. As part of our ongoing research to discover structurally interesting and biologically active metabolites from this strain, the MeOH extract of mycelia was further investigated, which led to the isolation of a new PKS-NRPS hybrid metabolite, pericoannosin B (1). Its structure and absolute configuration were determined on the basis of extensive spectroscopic data and circular dichroism (CD) spectral analyses. Herein, we report the isolation, structural elucidation, and biological activity of 1 (Fig. 1).

  图 1

  

  图 1  The structure of compound 1.

  Figure 1.  The structure of compound 1.

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  2.   Experimental

  2.1   General experimental procedures

  Optical rotations were recorded on a Perkin-Elmer Model-343 digital polarimeter. UV spectra were carried out on a V-650 spectrometer. CD spectra were measured on a JASCO J-815 spectropolarimeter. IR spectra were obtained on a Nicolet 5700 FT-IR microscope spectrometer. The ¹H NMR and ¹³C NMR spectra were carried out on VNS-600 spectrometer using acetone-d₆ as solvent and internal reference. Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in Hertz (Hz). ESI-MS data and HR-ESI-MS were obtained using an Agilent Technologies 6520 Accurate Mass Q-TOF LC/MS. Semi-preparative HPLC was performed on a Shimadzu LC-20AD instrument and a Shimadzu RID- 10A detector. A silica gel column (250 mm × 10 mm i.d., 5 μm; Apollo Silica, Alltech Co., Ltd., USA) was used for semi-preparative normal-phase HPLCand a C1°Column (250 mm × 10 mm i.d., 5 μm; Grace Adsorbosphere, W.R. Grace Co., USA) was used for semipreparative reversed-phase HPLC. Column chromatography (CC) was performed using Sephadex LH-20 and silica gel (200-300 mesh, Qingdao Marine Chemical Factory, China). Precoated silica gel GF₂₅₄ plates (Qingdao Marine Chemical Factory, China) were used for analytical TLC. Spots were detected on TLC under UV light or by heating after spraying with 10% H₂SO₄ in EtOH (v/v).

  2.2   Microorganism

  The fungal strain Periconia sp. F-31 was isolated from the leaves of A. muricata collected in Sanya district, Hainan province, China in September 2008, and identified using morphological and molecular (ITS1-5.8S-ITS2 rDNA sequence) analyses by China Pharmaceutical Culture Collection. The strain was deposited at the China Pharmaceutical Culture Collection (No. CPCC 400710), Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College.

  2.3   Fermentation, extraction and isolation

  The fungal strain was maintained on slants of modified potato dextrose broth (PDB) medium (potato 200 g, glucose 20 g, distilled water 1 L, KH₂PO₄ 3 g, MgSO₄ 0.73 g, vitamin B₁ 10 mg, pH 6.0-6.3; the media were autoclaved at 121 °C for 30 min) at 4 °C. The cultivation was performed in Erlenmeyer flasks (1000 mL) containing 300 mL of PDB liquid medium on a shaker at 130 rpm at 25 °C for 10 days. The cultures (140 L) were filtered under reduced pressure to afford the filtrate and mycelia.

  The mycelia were extracted with CH₃OH (4 × 3 L), and organic solvent was evaporated to dryness under vacuum to yield a crude extract (240 g). The CH₃OH extract was suspended in H₂O and partitioned using EtOAc to afford EtOAc extract (66 g). These extract was further separated by silica gel CC using CH₂Cl₂-acetone gradient (100:0-0:100) to give ten fractions on the basis of TLC analysis. Fraction Fm4 (2.7 g) was isolated by silica gel CC eluting with petroleum ether-acetone gradient (95:5-60:40) to yield six fractions (Fm4.1-4.6). Fraction Fm4.2 (510 mg) was separated by normal-phase semi-preparative HPLC eluting with n-hexane-isopropanol (25:1) at 4 mL/min to give four fractions (Fm4.2.1- 4.2.4). Fraction Fm4.2.2 (90 mg) was further purified by reversedphase semi-preparative HPLC eluting with CH₃CN-H₂O (67:33) at 4 mL/min to yield three fractions (Fm4.2.2.1-4.2.2.3). Fraction Fm4.2.2.2 (20 mg) was finally purified by normal-phase semipreparative HPLC eluting with n-hexane-EtOAc (7:1) at 4 mL/min to obtain 1 (3 mg, t_R = 13.6 min).

  Pericoannonsin B (1): White powder; [α]_D²⁰-85:3 (c 0.15, MeOH); IR (υ_max): 3300, 2957, 2927, 1680, 1441, and 1028 cm^-1; UV (MeOH) λ_max (logε): 203 (0.41) nm; CD (MeOH) ∆ε (nm): -5.66 (207), +0.99 (228.5), [Rh₂(OCOCF₃)₄]-induced CD ∆ε (nm): +0.28 (349.5); FABMS m/z 362.2 [M+H]⁺; HRESIMS m/z 384.2495 [M+Na]⁺ (calcd. for C₂₂H₃₅NNaO₃, 384.2509). ¹H NMR and ¹³C NMR data, see Table 1.

  表 1

  

  表 1  ¹H and ¹³C NMR spectral data of 1 and pericoannosin A in acetone-d₆.^a

  Table 1.  ¹H and ¹³C NMR spectral data of 1 and pericoannosin A in acetone-d₆.^a

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  2.4   Bioassays

  Cytotoxic activities were evaluated as previously reported [9]. Anti-HIV activities were evaluated as previously reported [11].

  3.   Results and discussion

  Pericoannosin B (1) was isolated as white powder and its molecular formula of C₂₂H₃₅NO₃ with six degrees of unsaturation was established by the HR-ESI-MS ion peak at m/z 384.2495 [M+Na]⁺. The IR spectrum displayed the presence of hydroxyl (3300 cm^-1) and carbonyl (1680 cm^-1) groups. The ¹³C NMR and DEPT spectra (Table 1) exhibited 22 carbon resonances, which consisted of four quaternary carbons (δ_C 179.0, 135.3, 133.7, and 98.1, including one carbonyl, two olefinic, one oxygenated), eight methines (δ_C 122.9, 120.9, 82.1, 51.2, 48.3, 38.7, 30.9, 25.7, including two olefinic, one oxygenated), five methylenes (δ_C 46.9, 40.8, 33.4, 33.0, 29.8), and five methyls (δ_C 23.9, 23.2, 22.6, 13.0, 11.2). Among the 22 carbons, one carbonyl carbon and two double bonds accounted for three degrees of unsaturation, suggesting that 1 possessed a tricyclic ring system. The HMBC correlations from H₂-4 to C-3, C-5, C-6 and C-10, from H₂-6 to C-7, C-8 and C-10, from H-7 to C-6, C-9 and C-21, from H-10 to C-5, C-6 and C-9, H-11 to C-3, C-5, C- 9 and C-10 indicated the presence of hexahydro-isochromen moiety. The HMBC cross-peaks from H-11 to C-12, C-13 and C-22, and from H-13 to C-11, C-14 and C-22 established one but-2-ene group connected to C-11 through C-12. Furthermore, the HMBC correlations from H-2 to C-1 and C-15, from H-15 to C-1, C-2, and C-16, together with the spin system from H-15 to H-20 on the basis of the ¹H-¹H COSY correlations, the degrees of unsaturation and the chemical shifts demonstrated the presence of five-membered lactam ring with an isobutyl group at C-16. In addition, the critical HMBC correlations from H-2 to C-3 and C-4, from 3-OH to C-2 supported the direct connection between C-2 and C-3. Thus, the planar structure of 1 was determined to be a hexahydro-1Hisochromen-5-isobutylpyrrolidin-2-one skeleton (Fig. 2), and identical to known compound, pericoannosin A [11], indicating that these two compounds were a pair of stereoisomers.

  图 2

  

  图 2  The key HMBC, ¹H–¹H COSY and NOESY correlations of compound 1.

  Figure 2.  The key HMBC, ¹H–¹H COSY and NOESY correlations of compound 1.

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  The relative configuration of 1 was determined by NOE experiments (Fig. 2). The NOEs of H-11/H-5 and 3-OH, H-6α/H-5 and H-4α, together with large coupling constants of ³J_{H-4β, H-5} (12.6 Hz) and ³J_{H-10, H-11} (10.2 Hz) and the small coupling constant of ³J_{H-4α, H-5} (3.6 Hz) suggested that H-4α, H-5, H-6α, H-11, and 3-OH were α-oriented, while H-4β and H-10 were β-oriented. The NOEs for H-2/H-15β, H-16/H-15α, along with large coupling constants of ³J_{H-2, H-15β} (9.6 Hz) and ³J_{H-16, H-15α} (7.8 Hz) and the small coupling constants of ³J_{H-2, H-15α} (8.4 Hz) and ³J_{H-16, H-15β} (3.6 Hz) indicated that H-2 and H-16 were on the opposite side of the lactam ring. The relative stereochemistry of C-2 and C-3 was not determined by NOE correlation between H-2 and 3-OH on account of short distance between H-2 and 3-OH. Detailed analysis of the structure revealed the presence of one hydroxyl connected to the chiral carbon (C-3) in 1, so the absolute configuration of C-3 can be determined by the CD data of the in situ-formed [Rh₂(OCOCF₃)₄] complex (Fig. S13) [11, 12]. After the addition of [Rh₂(OCOCF₃)₄] to a solution of 1 in CHCl₃, a metal complex was produced as an auxiliary chromophore. The Rh complex of 1 showed a positive E band (approximately 350 nm), suggesting the S configuration of C-3 by applying the bulkiness rule (Fig. S13 in Supporting information). According to CD octant rule, the chiral centre (C-2) near the carbonyl group of five-membered lactam ring play important role in generation of Cotton effect, so the Cotton effect around 210 nm of 1 was different from those of pericoannosin A (Fig. S14 in Supporting information) [11], indicating the R configuration of C-2. Therefore, the absolute configuration of 1 was determined to be 2R, 3S, 5S, 10R, 11R, 16S.

  Compound 1 was biologically assayed for cytotoxic (camptothecin as the positive control) and anti-HIV (efavirenz as the positive control) activities, unfortunately, it displayed low cytotoxic and anti-inflammatory activities with the IC₅₀ values >100 μmol/L.

  4.   Conclusion

  A new PKS-NRPS hybrid metabolite, pericoannosin B (1), was isolated from the endophytic fungus Periconia sp. F-31. This compound possessed uncommon hexahydro-1H-isochromen-5- isobutylpyrrolidin-2-one skeleton in natural resource, which was the stereoisomer as pericoannosin A. Pericoannosins A and B shared the same polyketide backbone, amino acid, as well as biosynthetic enzymes. Probably, the non-stereoselective Claisen reaction and hemiacetal reaction led to the pair of stereoisomers.

  Appendix A. Supplementary data

  Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.02.005.

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  Figure 1  The structure of compound 1.

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  Figure 2  The key HMBC, ¹H–¹H COSY and NOESY correlations of compound 1.

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  Table 1.  ¹H and ¹³C NMR spectral data of 1 and pericoannosin A in acetone-d₆.^a

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