Citrifelins A and B, Citrinin Adducts with a Tetracyclic Framework from

Aug 21, 2015 - Citrifelins A (1) and B (2), two citrinin adducts possessing a unique tetracyclic framework, were characterized from a coculture of mar...
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Citrifelins A and B, Citrinin Adducts with a Tetracyclic Framework from Cocultures of Marine-Derived Isolates of Penicillium citrinum and Beauveria felina Ling-Hong Meng,†,‡ Yang Liu,†,‡ Xiao-Ming Li,† Gang-Ming Xu,† Nai-Yun Ji,*,§ and Bin-Gui Wang*,† †

Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao 266071, People’s Republic of China ‡ University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, People’s Republic of China § Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Chunhui Road 17, Yantai 264003, People’s Republic of China S Supporting Information *

ABSTRACT: Citrifelins A (1) and B (2), two citrinin adducts possessing a unique tetracyclic framework, were characterized from a coculture of marine-derived fungal isolates of Penicillium citrinum and Beauveria felina. Neither fungus produced these compounds when cultured alone under the same conditions. The structures of these adducts were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assigned on the basis of TDDFTECD calculations. A hypothesis that adducts 1 and 2 might be derived from a citrinin derivative through a non-pericyclic Michael reaction is proposed. Compounds 1, 2, and 5 showed inhibitory activities against several human and aquatic pathogens.

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resulted in the isolation of two new citrinin derivatives, namely, citrifelins A and B (1 and 2), which possess a unique tetracyclic framework. This paper describes the isolation, structure determination, stereochemical assignment, and antimicrobial activity of compounds 1 and 2.

large number of structurally unique and biologically active secondary metabolites have been characterized from marine-derived microbes in the past two decades.1,2 However, finding new microbial natural products is becoming increasingly difficult, while the rate of reisolation of known secondary metabolites has significantly increased.3 It is well recognized that many microbial biosynthetic genes remain silent and are apparently not transcribed under a given set of standard laboratory conditions.4,5 To solve this problem, various strategies have been used to induce the production of new microbial secondary metabolites. One of these approaches is cocultivation of two or more microbial strains in one culture vessel, which sometimes induces the production of novel metabolites through activation and/or transcription of the silent biosynthetic genes.3,6 In order to investigate the chemical potential of fungi growing in the marine environment, we recently initiated a program for screening coculture fermentations of strains in our fungal library. Co-cultivation of two marine-derived fungi, Penicillium citrinum MA-197 and Beauveria felina EN-135, showed the production of several metabolites that were not produced when the two fungi were cultured alone (Figure S14, Supporting Information), and the extract of the coculture showed weak activity against the human pathogens Escherichia coli and Staphylococcus aureus. Several citrinin derivatives and cyclohexadepsipeptides have been isolated from P. citrinum MA-197 (Scheme S2, Supporting Information) and B. felina EN-135,7 respectively. A follow-up examination of the cocultivation of the two fungal strains © XXXX American Chemical Society and American Society of Pharmacognosy

The EtOAc extract of a coculture of P. citrinum MA-197 and B. felina EN-135 was purified by repeated column chromatogReceived: May 19, 2015

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DOI: 10.1021/acs.jnatprod.5b00450 J. Nat. Prod. XXXX, XXX, XXX−XXX

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raphy on silica gel, Sephadex LH-20, and Lobar LiChroprep RP-18, to yield citrifelins A and B (1 and 2). Citrifelin A (1) was determined to have the molecular formula C20H22O4 on the basis of positive ion HRESIMS, implying the presence of 10 degrees of unsaturation. The 13C NMR and DEPT data of 1 (Table 1) revealed the presence of three methyls, one aliphatic methylene, nine methines (five aromatic and three oxygenated aliphatic), and seven nonprotonated aromatic carbons. Table 1. NMR Data for Compounds 1 and 2 in CDCl3 1 no. 1 3 4 4a 5 6 7 8 8a 9 10 11 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 3′-OMe

δCa 63.3, CH 75.0, 35.1, 136.8, 113.8, 154.1, 100.5, 151.6, 113.8, 18.5, 21.7, 10.2, 133.9, 128.0, 115.6, 155.7, 115.6, 128.0, 78.1,

CH CH C C C CH C C CH3 CH3 CH3 C CH CH C CH CH CH

37.2, CH2

2 δH (J in Hz)b

4.88, dd (11.4, 3.8) 4.11, q (6.7) 2.64, q (6.8)

6.20, s

1.38, d (6.7) 1.21, d (6.8) 2.08, s 7.27, d (8.0) 6.81, d (8.0) 6.81, d (8.0) 7.27, d (8.0) 5.27, dd (12.1, 2.6) α 2.31, m β 2.10, m

δ Ca 63.6, CH 75.0, 35.1, 136.9, 114.1, 154.3, 100.5, 151.5, 112.1, 18.5, 22.4, 10.2, 133.5, 108.9, 146.9, 145.8, 114.5, 119.8, 78.4,

CH CH C C C CH C C CH3 CH3 CH3 C CH C C CH CH CH

37.2, CH2 56.2, CH3

δH (J in Hz)b 4.90, dd (11.4, 3.8) 4.12, q (6.5) 2.65, q (6.8)

Figure 1. Key COSY and HMBC correlations of 1 and 2.

6.21, s

1.39, d (6.5) 1.23, d (6.8) 2.08, s 6.92, s

6.88, d (6.8) 6.88, d (6.8) 5.26, dd (12.1, 2.6) α 2.30, m β 2.10, m 3.86, s

Figure 2. Key NOESY correlations observed for 1 (top) and 2 (bottom).

coupling constants for H-1 and H-8′β (11.4 Hz) as well as for H-7′ and H-8′β (12.1 Hz) indicated pseudo-diaxial relationships of the proton pairs. NOE correlations from H3-9 to H-4 and H-1 and from H-1 to H-7′ revealed the cofacial orientation of these groups, while an NOE cross-peak from H3-10 to H-3 placed these groups on the opposite face (Figure 2). To determine the absolute configuration, compound 1 was submitted to ECD calculation and measurement. A conformational search for 1 was performed via the Dreiding force field in MarvinSketch regardless of rotations of methyl and hydroxy groups,8 and the geometries were further optimized at the gasphase B3LYP/6-31G(d) level in Gaussian 09 to give one energy-minimized conformer (Figure S15) within a 3 kcal/mol energy threshold from the global minimum without vibrational imaginary frequencies.9 The optimized conformer was then subjected to calculation of its ECD spectrum using the timedependent density functional theory (TD-DFT) method at the gas-phase B3LYP/6-31G(d) level, using SpecDis software.10 Comparison of the experimental and calculated spectra for 1 showed excellent agreement (Figure 3) for the 1R, 3R, 4S, 7′Rabsolute configuration in 1. Both spectra presented a strong positive Cotton effect (CE) below 225 nm, a strong negative CE near 240 nm, and a weak positive CE around 270 nm (Figure 3), and these close similarities enabled assignment of the absolute configuration of 1 as shown. Citrifelin B (2) was assigned the molecular formula C21H24O5, with one OCH2 unit more than 1, on the basis of HRESIMS data. Its NMR spectroscopic data were very similar

a

Measured at 125 MHz and multiplicities were determined by DEPT and HSQC. bMeasured at 500 MHz.

The 1H NMR spectrum of 1 showed well-dispersed signals (Table 1 and Figure S1), and, aided by the HSQC experiment, these signals were revealed to represent three methyls (one singlet and two doublets), one aliphatic methylene, and nine methines (five aromatic and three oxygenated aliphatic), which agreed with the 13C NMR data. Detailed interpretation of the COSY spectrum of 1 led to the elucidation of three discrete proton−proton spin-coupling systems corresponding to a CH3−CH(O)−CH−CH3 moiety (I, C-9, C-3, C-4, and C-10), a −CH(O)−CH2−CH(O)− unit (II, C-1 to C-8′ and C-7′), and an oxygenated, p-disubstituted aromatic ring (Figure 1). HMBC correlations from H-1 to C-3, C-8, and C-8a, from H-7′ to C-1 and C-8, from H-8′ to C-8a, from H-7 to C-5 and C-8a, from H3-11 to C-4a and C-6, from H3-10 to C-3, and from H3-9 to C-4 not only connected units I and II but also resulted in elucidation of the substructure comprising rings A, B, and C in 1 (Figure 1). Further HMBC correlations from H-8′ to C-1′ and from H-7′ to C-2′/C-6′ verified the linkage of the p-hydroxyphenyl group at C-7′. The relative configuration of 1 was deduced from 1H−1H coupling constants and NOESY data (Figure 2). The coupling constant between H-3 and H-4 (0 Hz) suggested a diequatorial or equatorial−axial orientation of H-3 and H-4, while the large B

DOI: 10.1021/acs.jnatprod.5b00450 J. Nat. Prod. XXXX, XXX, XXX−XXX

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act as a nucleophile (a Michael donor) in a mildly basic environment. In this hypothesis, the acetophenone precursor (1-(4-hydroxyphenyl)ethanone for 1 or 1-(4-hydroxy-3methoxyphenyl)ethanone for 2) could add to the double bond of the decarboxycitrinin tautomer, followed by an attack on the acetophenone carbonyl to generate the heterocyclized intermediate. Compounds 1 and 2 could be finally formed from the heterocyclic intermediate by dehydration and reduction (Scheme 1). To support this hypothesis, however, further experimental evidence is necessary. The isolated compounds were submitted for antimicrobial screening,16 and compound 1 showed inhibitory activity against the human pathogens E. coli and S. aureus with an MIC value of 8.0 μg/mL for each bacterium, with compound 2 having MIC values of 2.0 and 4.0 μg/mL, respectively, indicating that the presence of the OMe at C-3′ likely strengthens the activity. Compounds 1−9 were also assayed for activities against the aquatic pathogens Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus, V. anguillarum, V. harveyi, and V. parahemolyticus. No strong activity was observed, but compounds 1, 2, and 5 showed weak activity against V. alginolyticus, with MIC values of 16.0, 16.0, and 8.0 μg/mL, respectively. In conclusion, we isolated and identified two new citrinin derivatives (1 and 2) with a unique tetracyclic framework from the coculture broth of two marine-derived fungi, P. citrinum MA-197 and B. felina EN-135, whereas neither fungus could produce these compounds when cultured alone. The fact that cocultivation in the present study induced the production of new fungal metabolites demonstrates the general value of such cocultivating experiments and encourages future studies.

Figure 3. Experimental and calculated ECD spectra of 1.

to those of 1, except for the replacement of the p-disubstituted aromatic ring signals with those characteristic of a 1,2,4trisubstituted benzene, together with addition of signals for a methoxy group resonating at δH 3.86/δC 56.2 (3′-OMe) and a nonprotonated carbon at δC 146.9 (C-3′). These data suggested that the 4-hydroxyphenyl group in 1 was replaced by a 3-methoxy-4-hydroxyphenyl (or 4-hydroxy-3-methoxyphenyl) unit in 2. This deduction was supported by COSY and HMBC correlations (Figure 1), with the latter establishing the location of the methoxy group. The relative configuration for 2 was shown to be the same as that of 1, as evidenced by the similar coupling patterns of the two compounds (Table 1) as well as by correlations from H3-9 to H-4 and H-1 and from H-1 to H-7′ in the NOESY spectrum of 2 (Figure 2). The ECD spectrum of 2 showed a negative CE at 242 nm and two positive CEs at 222 and 287 nm, which was quite similar to that of 1. Thus, the absolute configuration of 2 was assigned to match that of 1. In addition to compounds 1 and 2, three known citrinin derivatives, namely, dicitrinin A (3),11 dihydrocitrinone (4),12 and decarboxydihydrocitrinone (5),12 as well as four known cyclohexapeptides, destruxin A,13 roseotoxin B,14 and isaridins A and B,15 were also isolated and identified from the coculture extract. A number of natural occurring citrinin adducts have been reported, and some of them are described to be derived from citrinin through a Diels−Alder adduct reaction with a range of dienophiles.11,12 However, no solid experimental support has yet been published. In addition to a Diels−Alder reaction, we herein hypothesize that compounds 1 and 2 might be derived from a citrinin derivative through a Michael reaction with an acetophenone derivative (as its enolate tautomer), which could

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on an Optical Activity AA-55 polarimeter. UV spectra were measured on a PuXi TU-1810 UV−visible spectrophotometer. ECD spectra were acquired on a Chirascan spectropolarimeter. Quantum chemical calculations were conducted using Gaussian 09 software. IR data were obtained on a Thermo Scientific Nicolet iN10 spectrophotometer. NMR spectra were recorded on a Bruker Avance 500 spectrometer with TMS as an internal standard. Mass spectra were determined on a VG Autospec 3000 or an API QSTAR Pulsar 1 mass spectrometer. HPLC was performed using a Dionex HPLC system equipped with a P680 pump, an ASI-100 automated sample injector, and a UVD340U multiple-wavelength detector controlled by Chromeleon software (version 6.80). Commercially available Si gel (200−300 mesh, Qingdao Haiyang Chemical Co.), Lobar LiChroprep RP-18 (40−63 μm, Merck), and Sephadex LH-20 (Pharmacia) were used for open-column chromatography. Solvents for extraction and purification were distilled prior to use.

Scheme 1. Proposed Biosynthetic Pathway for 1 and 2

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DOI: 10.1021/acs.jnatprod.5b00450 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Fungal Material. The isolation and identification of the fungus B. felina EN-135 were described previously.7 P. citrinum MA-197 was isolated from the mangrove plant Lumnitzera racemosa and was identified using the protocol in our previous report.17 The resulting sequence data obtained from the fungal strain MA-197 have been deposited in GenBank (with accession no. KP279928). A BLAST search result indicated that the sequence was most similar (99%) to the sequence of P. citrinum (compared to no. KM491892.1). Both strains EN-135 and MA-197 are preserved at the Key Laboratory of Experimental Marine Biology, Institute of Oceanology of the Chinese Academy of Sciences (IOCAS). Fermentation, Extraction, and Isolation. P. citrinum MA-197 and B. felina EN-135 were each grown on PDA medium at 28 °C for 4 days, and B. felina EN-135 was then inoculated into 1 L conical flasks (20 × 100 mL, a total of 2 L) each containing 100 mL of wheat bran broth medium (100 mL of naturally sourced and filtered seawater from the Huiquan gulf of the Yellow Sea near the campus of IOCAS, 100 g of wheat bran, and 0.6 g of dried potato powder) at room temperature. After 3 days, a full loop of P. citrinum MA-197 was transferred aseptically to each flask culture of B. felina EN-135 and reincubated at room temperature for 28 days. As a control, a full loop of P. citrinum MA-197 was transferred aseptically to 1 L conical flasks (10 × 100 mL, a total of 1 L) of wheat bran broth medium and incubated under the same conditions for 28 days. The fermented cocultures were exhaustively extracted with EtOAc (150 mL/flask). The combined EtOAc solution was concentrated under reduced pressure to give an extract (18 g), which was fractionated by silica gel vacuum liquid chromatography (VLC) using different solvents of increasing polarity from petroleum ether (PE) to MeOH to yield seven fractions (Frs. 1−7) based on TLC analysis. Fr. 4 (2.1 g) was further purified by reversed-phase column chromatography (CC) over Lobar LiChroprep RP-18 with a MeOH−H2O gradient (from 20:80 to 100:0) to afford three subfractions (Fr. 4-1 to Fr. 4-3). Fr. 4-2 (86 mg) was further purified by CC on silica gel eluting with a CHCl3−MeOH gradient (from 120:1 to 50:1) and then by Sephadex LH-20 (MeOH) to afford compounds 1 (1.9 mg) and 2 (8.1 mg). The fermented monoculture of P. citrinum MA-197 was exhaustively extracted with EtOAc (150 mL/flask). The combined EtOAc solution was concentrated under reduced pressure to give an extract (10 g), which was fractionated by VLC using different solvents of increasing polarity from PE to MeOH to yield five fractions (Frs. 1−5) based on TLC analysis. Details of the isolation and purification of other compounds from coculture together with the extraction and isolation of compounds from the monoculture of P. citrinum MA-197 are provided in the Supporting Information. As for the metabolites of B. felina EN-135, cyclohexadepsipeptide derivatives were found to be the major components when it was cultured alone, as reported in our previous chemical investigations.7 Citrifelin A (1): yellow solid; [α]20D +40 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 208 (4.62), 281 (3.61) nm; ECD (0.49 mM, MeOH) λmax (Δε) 221 (0.93), 242 (−1.46), 273 (0.35) nm; 1H and 13 C NMR data, Table 1; HRESIMS m/z 327.1592 [M + H]+ (calcd for C20H23O4, 327.1591). Citrifelin B (2): yellow solid; [α]20D +35 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 206 (4.66), 281 (3.72) nm; IR νmax 3446, 2968, 2930, 2867, 1608, 1518, 1455, 1382, 1278, 1120 cm−1; ECD (0.45 mM, MeOH) λmax (Δε) 222 (2.05), 242 (−2.67), 287 (0.40) nm; 1H and 13C NMR data, Table 1; HRESIMS m/z 357.1699 [M + H]+ (calcd for C21H25O5, 357.1697). Antimicrobial Assay. Antimicrobial assays against E. coli, S. aureus, A. hydrophilia, V. parahemolyticus, V. harveyi, E. tarda, V. alginolyticus, and V. anguillarum were carried out using the well diffusion method,16 and chloromycetin was used as a positive control.

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Details of the isolation and purification of compounds from the cocultures and from the monocultures of P. citrinum MA-197 as well as their chemical structures, selected 1D and 2D NMR spectra of compounds 1 and 2, and the HPLC profiles of extracts from B. felina, P. citrinum, and a representative coculture (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-535-2109176. Fax: +86-535-2109000. E-mail: nyji@ yic.ac.cn (N.-Y. Ji). *Tel and Fax: +86-532-82898553. E-mail: [email protected]. cn (B.-G. Wang). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (NSFC grant no. 31270104) and from the NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402) is gratefully acknowledged.

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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.jnatprod.5b00450. D

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(15) Ravindra, G.; Ranganayaki, R. S.; Raghothama, S.; Sri nivasan, M. C.; Gilardi, R. D.; Karle, I. L.; Balaram, P. Chem. Biodiversity 2004, 1, 489−504. (16) Al-Burtamani, S. K. S.; Fatope, M. O.; Marwah, R. G.; Onifade, A. K.; Al-Saidi, S. H. J. Ethnopharmacol. 2005, 96, 107−112. (17) Wang, S.; Li, X. M.; Teuscher, F.; Li, D. L.; Diesel, A.; Ebel, R.; Proksch, P.; Wang, B. G. J. Nat. Prod. 2006, 69, 1622−1625.

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