Lumazine Peptides Penilumamides B–D and the Cyclic Pentapeptide

Article pubs.acs.org/jnp

Lumazine Peptides Penilumamides B−D and the Cyclic Pentapeptide Asperpeptide A from a Gorgonian-Derived Aspergillus sp. Fungus Min Chen,† Chang-Lun Shao,† Xiu-Mei Fu,† Chui-Jian Kong,† Zhi-Gang She,‡ and Chang-Yun Wang*,†,§ †

Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People’s Republic of China ‡ School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China § Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, People’s Republic of China S Supporting Information *

ABSTRACT: Three new lumazine peptides, penilumamides B−D (2−4), and one known analogue, penilumamide (1), together with a new cyclic pentapeptide, asperpeptide A (5), were isolated from the gorgonian-derived fungus Aspergillus sp. XS-20090B15. Among them, 2 was obtained from the feeding culture with L-methionine of this strain. All structures were elucidated by spectroscopic methods and chemical derivatization. Compounds 1−4 are rare lumazine peptides, of which 1 and 3 are formed from 2 by oxidation of the L-methionine residue.

L

of these peptides. An incubation experiment where methionine was added to this strain is also described.

inear and cyclic peptides have attracted great attention in recent years for their unique structures and potent pharmacological activities, many of which have played a significant role in pharmaceutical research as useful agents or as lead compounds for drug development.1 Marine-derived fungi, especially those belonging to the genera Aspergillus and Penicillium, have proven to be an excellent source of novel peptides.2 For example, penilumamide, a lumazine peptide from a red algal derived Penicillium sp. fungus,3 is the first and hitherto only report of a linear peptide with an unusual 1,3dimethyllumazine-6-carboxylic acid starter unit. Although lumazines are well-known metabolites and are frequently present in some synthetic molecules, natural products with an N-methylated lumazine are relatively rare, occasionally occurring in marine invertebrates or freshwater leeches.4 In the course of our ongoing investigation on new secondary metabolites from marine-derived fungi,5 the fungal strain Aspergillus sp. XS-20090B15, isolated from the inner part of a fresh Muricella abnormaliz gorgonian, attracted our attention because the culture extract exhibited a distinct TLC profile and characteristic UV absorption spectra observed by HPLC-UV. Chemical investigation of the normal culture and an Lmethionine supplemented culture resulted in the isolation of four linear peptides (1−4) and one cyclic peptide (5). All of the linear peptides contain a rare 1,3-dimethyllumazine-6carboxylic acid starter unit. More specifically, three of these lumazine peptides possess a methionine, a methionine sulfoxide, or a methionine sulfone residue. Herein, we report the isolation, structural characterization, and biological activities © XXXX American Chemical Society and American Society of Pharmacognosy

L-

■

RESULTS AND DISCUSSION The gorgonian-derived fungus Aspergillus sp. XS-20090B15 was first cultivated in normal potato glucose liquid medium (30 L) at 20 °C without shaking for 5 weeks. The culture extract showed a characteristic TLC profile (pale yellow spots, colored by anisaldehyde/sulfuric acid indicator) and HPLC-UV absorption spectra (λmax = 223, 250, 319 nm), which implied a group of analogues structurally closely related to penilumamide (1), a lumazine peptide reported very recently from a Penicillium sp. fungus.3 Four compounds were isolated from the broth and mycelia, including the major lumazine peptide penilumamide (1) and two minor analogues, penilumamides C and D (3 and 4), together with one cyclic peptide, asperpeptide A (5). All of the linear lumazine peptides (1, 3, and 4) contain an N-methylated lumazine moiety, which is rare in natural products. To date, 1 is the only reported lumazine peptide. Interestingly, 1 and 3 are lumazine peptides with a methionine sulfoxide or a methionine sulfone residue. A literature survey revealed that the methionine sulfoxide or sulfone derivatives were most probably formed by oxidation of the methioninecontaining natural products during isolation.6 In the present study, it could be speculated that both 1 and 3 likely arise from the corresponding lumazine peptide bearing a methionine Received: February 22, 2014

A

dx.doi.org/10.1021/np5001686 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Penilumamide C (3) was also isolated as a white powder. The molecular formula C22H24N6O8S of 3 revealed one oxygen atom more than that of 1 and two oxygen atoms more than that of 2. The most obvious difference in the 1H NMR spectrum (Table 1) was the downfield shifts of H-7′ (δH 2.96) and H-5′ (δH 3.22−3.34) neighboring the sulfur atom in 3 compared to those of 1 and 2, indicating a methionine sulfone residue in 3 instead of the methionine sulfoxide residue in 1 and methionine residue in 2. The ESIMS/MS analysis for the sodium adduct [M + Na]+ (m/z 555) further confirmed the planar structure of 3 (Figure 2). The absolute configuration of 3 was determined based on chemical transformation from 1. Compound 3 can be obtained by oxidation of 1 in a mixture of CH2Cl2, glacial HOAc, and 30% aqueous H2O2 at room temperature (rt) for 3 h. Thus, the absolute configuration of the methionine sulfone residue in 3 was assigned as L, the same as the configuration of the methionine sulfoxide residue in 1. Compound 2 was obtained from the methionine-rich fermentation broth, together with its oxidative products 1 and 3, suggesting that L-methionine might be the precursor of 1−3. Interestingly, the yields of 2 and its primary oxidative product 1 were found to increase notably in response to the concentration of the L-methionine, indicating that L-methionine may play an important role in the biosynthesis of lumazine peptides. It should be pointed out that 1 and 2 were unstable when exposed to air. When 2 was exposed to air at rt, 1 was detected after a few days and 3 appeared several days later. Therefore, the absence of 2 from the normal culture medium is not surprising due to the small amount formed and efficient oxidation of 2 into 1 and 3 during the whole process of fermentation and isolation. Penilumamide D (4) was also isolated as a white powder with the molecular formula of C19H19N7O5. Its 1H and 13C NMR spectra (Table 1) were very close to those of 2 except for a methyl signal [δC‑4′ 17.7, δH‑4′ 1.53 (d, J = 7.2 Hz)] and a methine signal [δC‑2′ 50.6, δH‑2′ 4.65 (m)] in 4 instead of the corresponding methionine signals of 2, thus revealing an alanine residue was contained in 4. The COSY cross-peaks of 1′-NH/H-2′/H-4′ and the HMBC correlations from H-4′ to C2′ and C-3′ and from H-2′ to C-3′ and C-4′ confirmed the alanine residue in 4 (Figure 1). In addition, the carbon signal at δC‑8″ 171.0 and the proton signals at δH‑9″ 7.56 and 8.19 (two protons bonded to a nitrogen atom) suggested that an amide in 4 replaced the corresponding methyl ester in 2. The HMBC correlations from 9″-NH2 to C-3″ and from H-4″ to C-8″ confirmed the amide group anchored at C-3″ of the benzene ring in 4. Thus, 4 was established as a peptide with a 1,3dimethyllumazine-6-carboxylic acid, coupled to an alanine and an anthranilic amide. The planar structure of 4 was further confirmed by the ESIMS/MS analysis for the sodium adduct [M + Na]+ (m/z 448) (Figure 2). The Marfey’s analysis showed the presence of an L-alanine residue in 4 (Figure S32). Asperpeptide A (5) was isolated as a white powder. Its molecular formula was determined to be C27H31N5O7 based on HRESIMS, requiring 15 degrees of unsaturation. Analysis of the 1 H and 13C NMR spectra (Table 2) of 5 indicated the presence of five carbonyl signals and four amide protons, suggesting a pentapeptide structure for 5. Detailed interpretation of the COSY and HMBC spectra of 5 constructed the individual amino acids as one tyrosine, two alanines, one proline, and one 5-hydroxyanthranilic acid. These residues accounted for 14 of the 15 degrees of unsaturation, indicating that 5 is a cyclic peptide. The sequence and connectivity of the amino acid

residue. This putative lumazine peptide might be oxidized easily during the extraction and purification procedures or even in the fermentation process. Furthermore, L-methionine likely is involved in the biogenic synthesis of this putative lumazine peptide. On the basis of the above considerations, fermentation experiments including L-methionine were designed to try to get enough of the putative lumazine peptide. To avoid the oxidation of the putative lumazine peptide, the extraction and purification were performed immediately after harvesting the fungal cultures. As expected, a new chromatographic peak (tR = 46.8 min; UV: λmax = 220, 252, 317 nm) appeared in the HPLC profile of the supplemented culture compared to the normal culture (Figure S34). Chemical investigation of this new chromatographic peak from the methionine-rich fermentation culture led to the isolation of penilumamide B (2), a new lumazine peptide possessing a methionine residue.

Penilumamide B (2) was obtained as a white powder. Its molecular formula, C22H24N6O6S, was determined on the basis of HRESIMS. Careful comparison of the NMR spectra indicated that 2 was very similar to penilumamide (1) (Table 1).3 In the 1H NMR spectrum, the significant upfield chemical shifts of the methylene (2: δH‑5′ 2.66−2.69 vs 1: δH‑5′ 2.86− 2.97) and the methyl (2: δH‑7′ 2.15 vs 1: δH‑7′ 2.58) attached to the sulfur atom in 2 suggested a methionine moiety in 2 instead of the methionine sulfoxide moiety in 1. Detailed analysis of the 2D NMR experiments (Figure 1) unambiguously assigned the methionine moiety for 2. To further confirm the planar structure of 2, its protonated molecule [M + H]+ (m/z 501) was subjected to ESIMS/MS experiments (Figure 2). The fragments, including elimination of a mercaptomethyl group, demethoxylation of the ester group, or cleavage of the peptide bond bound to the benzene ring system, supported the planar structure of 2 as shown. The absolute configuration of the methionine residue in 2 was determined by Marfey’s method. HPLC analysis of the FDAA derivative of the hydrolysate from 2 gave the same retention time as the derivative prepared from authentic L-methionine (Figure S31), and therefore, the Lconfiguration was assigned to the methionine residue in 2. B

dx.doi.org/10.1021/np5001686 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Spectroscopic Data for 2−4 at 600 (1H) and 150 (13C) MHz 2a position 2 4 4a 6 7 8a 9 10 11 1′-NH 2′ 3′ 4′

a

δC, type 150.4, 159.4, 125.1, 140.1, 148.1, 149.6, 29.3, 29.9, 162.6,

C C C C CH C CH3 CH3 C

52.4, CH 169.6, C 31.3, CH2

5′

30.4, CH2

7′ 1″-NH 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″

30.4, CH3 140.8, 115.6, 120.6, 134.7, 123.1, 130.9, 168.5, 54.2,

C C CH CH CH CH C CH3

3a

δH, mult. (J in Hz)

9.44, s 3.76, s 3.55, s 8.79, d (8.4) 5.00, dt (8.4, 4.8) 2.44−2.47, m 2.25−2.28, m 2.66−2.69, m 2.15, s 11.55, s

8.67, 7.53, 7.09, 7.98,

dd (8.4, 1.8) dt (8.4, 1.8) dt (8.4, 1.8) dd (8.4, 1.8)

3.78, s

δC, type 150.3, 159.4, 125.2, 139.7, 148.1, 149.4, 29.3, 29.9, 162.8,

δH, mult. (J in Hz)

C C C C CH C CH3 CH3 C

53.3, CH 168.5, C 25.3, CH2 51.4, CH2 40.7, CH3 140.4, 115.7, 120.5, 134.7, 123.4, 130.9, 168.4, 52.4,

4b

C C CH CH CH CH C CH3

9.42, s 3.76, s 3.53, s 8.99, d (8.4) 5.00, dt (8.4, 5.4) 2.68−2.74, 2.47−2.53, 3.22−3.27, 3.29−3.34, 2.96, s 11.58, s

8.62, 7.53, 7.10, 7.97,

m m m m

50.6, CH 171.0, C 17.7, CH3

δH, mult. (J in Hz)

9.29, s 3.60, s 3.36, s 9.07, d (7.2) 4.65, m 1.53, d (7.2)

12.10, s

dd (8.4, 1.8) dt (8.4, 1.8) dt (8.4, 1.8) dd (8.4, 1.8)

3.77, s

δC, type 150.9, C 159.7, C 126.7, C 139.8, C 147.2, CH 149.9, C 29.2, CH3 29.9, CH3 163.0, C

140.1, 120.3, 120.5, 132.7, 123.1, 129.1, 171.0,

C C CH CH CH CH C

8.49, 7.49, 7.12, 7.77,

dd (7.8, 1.8) dt (7.8, 1.8) dt (7.8, 1.8) dd (7.8, 1.8)

7.56, brs 8.19, brs

Recorded in CDCl3. bRecorded in DMSO-d6.

Figure 1. COSY and key HMBC correlations for 2, 4, and 5.

deuterated solvents, DMSO-d6 and acetone-d6. The NOE correlations in two deuterated solvents were almost the same, suggesting that the conformation of 5 is relatively stabilized in solution. In addition, the Pro-Ala amide bond adopts a trans conformation by the small chemical shift difference (5.1 ppm) between the β- and γ-carbons of Pro observed in the 13C NMR spectrum of 5 (Δδβγ: trans < 6 ppm, cis > 8 ppm).7 A literature search revealed that lumazine peptide penilumamide (1) showed no cytotoxic and antimicrobial activities and no influence on the Ca2+ level.3 In the present study, besides the cytotoxic and antibacterial activities, all of the isolated compounds were assessed for their antiviral and acetylcholinesterase inhibitory activities. Unfortunately, no activity was found for the linear lumazine peptides (1−4). The cyclic pentapeptide (5) exhibited antibacterial activity against Bacillus cereus and Staphylococcus epidermidis with the same MIC value of 12.5 μM.

residues were deduced by HMBC experiments and ESIMS/MS analysis. The HMBC correlations from the NH protons of the amino acid residues to the carbonyl carbons of the neighboring residues suggested two partial amino acid sequences, 5-OHAAPro and Ala-Ala-Tyr (Figure 1). The complete cyclic sequence was unambiguously determined by ESIMS/MS experiments for the sodium adduct [M + Na]+ (m/z 560) (Figure 2). Ring opening occurred at the Tyr-5-OHAA amide bond and gave a series of key ion peaks corresponding to the proposed interpretations in the ESIMS/MS spectrum, establishing the sequence for 5 as cyclo(-Pro-Ala-Ala-Tyr-5-OHAA). Marfey’s method was employed to determine the absolute configurations of the amino acid residues in 5 (Figure S33). HPLC analysis of the FDAA derivatives of the hydrolysate from 5 showed that all of the amino acid residues possessed the L-configurations. The conformations of cyclic peptides are constrained and often stabilized by intramolecular hydrogen bonds. The 2D NOESY experiments of 5 were carried out in two different C

dx.doi.org/10.1021/np5001686 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 2. Key ESIMS/MS fragments for 2−5.

Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for 5 in DMSO-d6 amino acid proline

alanine

alanine

tyrosine

5-OH-anthranilic acid

position

δC

1 2 3

170.3, C 60.2, CH 29.9, CH2

4 5

24.8, CH2 50.8, CH2

6 7 8 NH 9 10 11 NH 12 13 14

172.0, C 48.9, CH 17.6, CH3

15 16/20 17/19 18 NH 21 22 23 24 25 26 27 NH

129.3, 130.5, 115.6, 156.4,

C CH CH C

168.1, 128.7, 113.3, 153.9, 117.2, 123.0, 126.4,

C C CH C CH CH C

170.2, C 49.5, CH 17.7, CH3 173.7, C 56.9, CH 33.6, CH

Aspergillus sp. XS-20090B15. Among them, 2−4 represent the second examples of lumazine peptides with a 1,3-dimethyllumazine-6-carboxylic acid moiety, which is rare in natural occurring peptides. Peptides possessing methionine are relatively uncommon, while their methionine sulfone or sulfoxide derivatives have been reported frequently. In the present study, the lumazine peptide 2, possessing a methionine residue, was obtained through a feeding experiment with Lmethionine, suggesting that L-methionine is a precursor of 1−3.

δH (J in Hz) 4.38, 2.05, 1.63, 1.82, 3.48, 3.16,

m m m m m m

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Nicolet-Nexus-470 spectrometer using KBr pellets. UV spectra were obtained on a Beckman DU 640 spectrophotometer. NMR spectra were acquired on a JEOL JEM-ECP NMR spectrometer (600 MHz for 1H and 150 MHz for 13C), using TMS as an internal standard. ESIMS spectra were measured on a Micromass Q-TOF spectrometer. Semipreparative HPLC was performed on a Waters 1525 system coupled with a Waters 2996 photodiode array detector, using a C18 (Kromasil, 5 μm, 250 × 10 mm2) column for semipreparative HPLC and a CHI-TBB column (Kromasil, 5 μm, 150 × 4.6 mm) for chiral-HPLC. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200−300 mesh), Sephadex LH-20 (Amersham Biosciences), and octadecylsilyl silica gel (Unicorn; 45−60 μm) were used for column chromatography. Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin-layer chromatography. Isolation of the Fungal Material. The fungus Aspergillus sp. XS20090B15 was isolated from the inner part of a fresh Muricella abnormaliz gorgonian, which was collected from the Xisha Islands coral reef of the South China Sea in December 2009. The strain was deposited at the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, People’s Republic of China. Following surface sterilization with 75% EtOH for 30 s, the gorgonian was rinsed three times in sterile H2O. To distinguish the remaining epiphytic fungi from endophytic fungi, an imprint of the gorgonian surface on PDA was made. Small tissue samples from the inner part of the gorgonian were aseptically cut and pressed onto PDA plates containing an antibiotic to suppress bacterial growth (composition of isolation medium: potatoes 200 g/L, glucose 20 g/L, agar 15 g/L,

4.34, m 1.32, d (7.2) 7.30, d (8.4) 4.36, m 1.33, d (7.2) 7.61, d (8.4) 3.96, m 3.26, dd (13.8, 4.2) 3.05, dd (13.8, 12.0) 6.96, d (8.4) 6.68, d (8.4) 9.02, d (7.2)

6.79, d (2.4) 6.84, dd (8.4, 2.4) 7.83, d (8.4) 8.70, s

In conclusion, three new lumazine peptides, penilumamides B−D (2−4), and a new cyclic pentapeptide, asperpeptide A (5), were isolated from the gorgonian-derived fungus D

dx.doi.org/10.1021/np5001686 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Asperpeptide A (5): white powder; [α]25D −10 (c 0.10, CH2Cl2); UV (MeOH) λmax (log ε) 225 (2.40), 255 (1.20), 306 (0.24) nm; IR (KBr) νmax 3365, 2944, 2832, 1665, 1598, 1516, 1453, 1240, 1027, 637 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Table 2; HRESIMS m/z 560.2126 [M + Na]+ (calcd for C27H31N5O7Na, 560.2116). Marfey’s Analysis of 2, 4, and 5 (ref 8). Under an argon atmosphere, a solution of 2 (4 or 5, 1 mg) in 6 N HCl (1 mL) was heated at 100 °C for 12 h. The excess HCl was removed under vacuum. Then 250 μL of H2O was added to dissolve the hydrolysate, and 100 μL of the solution was placed in a 1 mL reaction vial and treated with a 1% solution of FDAA (200 μL) in acetone followed by 1.0 M NaHCO3 (40 μL). The reaction mixture was heated at 45 °C for 1.0 h and cooled. The mixture was acidified with 2.0 M HCl (20 μL). Separately, the standard amino acids L-Met and D-Met (L-Ala, DAla, L-Tyr, D-Tyr, L-Pro, D-Pro) were derivatized with FDAA in the same manner as that of 2 (4 or 5). All FDAA derivatives were analyzed by HPLC using MeCN−H2O (0.2% TFA) as the mobile phase (see Supporting Information). Oxidation Conversion of 1. Compound 1 (1 mg) was dissolved in 1 mL of CH2Cl2, and 1 mL of glacial HOAc was added. After cooling at 4 °C for 10 min, 50 μL of 30% aqueous H2O2 was added to the reaction mixture, which was stirred at rt for 3 h. The solvents were evaporated to dryness to obtain the semisynthetic product 3. The TLC and HPLC analysis of the semisynthetic sample showed an Rf value identical to that of the authentic sample. Co-injection of these two samples on a chiral phase column (88% n-hexane−2-propanol, tR = 22.1 min) confirmed their identity. Biological Assays. The antibacterial assay was carried out as described previously.9 The 10 bacterial strains Bacillus cereus, Salmonella enterica, Kocuria rhizophila, Staphylococcus epidermidis, S. aureus, Nocardia brasiliensis, Escherichia coli, Vibrio parahemolyticus, Pseudomonas aeruginosa, and P. putida were used, and ciprofloxacin was used as a positive control. Cytotoxicities against the human erythroleukemia K562 and human promyelocytic leukemia HL-60 cell lines were determined by the MTT method,10 and cytotoxicities against the human lung carcinoma cell line A549, human colorectal cancer cell line HCT-116, and human cervical carcinoma cell line HeLa were measured by the SRB assay.11 Adriamycin was used as a positive control. Antiviral activity was tested against respiratory syncytial virus (RSV) and herpes simplex virus (HSV-1) according to established procedures.12 Ribavirin was used as a positive control. The AChE inhibitory activity was determined using Ellman’s method.13 Huperzine A was used as a positive control.

chloramphenicol 0.2 g/L, and natural sea salt (from Yangkou saltern, China) 12 g/L in H2O, pH 7.4−7.8). After incubation at 28 °C, the fungal strain under investigation was found to grow exclusively out of the gorgonian tissue, but not on the agar plates taken from the imprint of the gorgonian surface. A pure strain of Aspergillus sp. XS-20090B15 was isolated from the growing cultures by repeated reinoculation on PDA plates. Identification of Fungal Cultures. The fungus was identified as Aspergillus sp. according to its morphological traits and a molecular protocol by amplification and sequencing of the DNA sequences of the ITS region of the rRNA gene as described previously.5d The fungus was identified as an Aspergillus sp. whose 566 base pair ITS sequence had 100% sequence identity to that of Aspergillus sp. HZ-35 (EU301661). The sequence data have been submitted to GenBank with accession number HM991281. Fermentation, Extraction, and Isolation. The fungus Aspergillus sp. XS-20090B15 was first cultivated in normal potato glucose liquid medium (20 g of glucose and 12 g of natural sea salt (from Yangkou saltern, China) in 1 L of potato infusion; 1 L Erlenmeyer flasks each containing 300 mL of culture broth) at 20 °C without shaking for 5 weeks. The culture (30 L) was filtered to separate the broth from the mycelia. Then the broth was extracted three times with an equal volume of EtOAc, and the mycelia were extracted three times with MeOH. The organic extracts were combined and concentrated under vacuum to afford a total extract (broth extract and mycelia extract, 27.8 g), which was separated into seven fractions (Fr.1−Fr.7) by silica gel column chromatography (CC) using a step gradient elution with MeOH−CH2Cl2 (0−100%). Fr.4 was separated by silica gel CC eluting with CH2Cl2−MeOH (v/v, 30:1) to give 1 (82 mg) and 3 (7.5 mg). Fr.5 was subjected to Sephadex LH-20 CC eluting with a mixture of CH2Cl2−MeOH (v/v, 1:1) and then was further purified by using semipreparative HPLC (75% MeOH−H2O) to obtain 4 (6.5 mg). Fr.6 was subjected to an ODS column eluting with 70% MeOH−H2O and finally was purified by using semipreparative HPLC (65% MeOH− H2O) to yield 5 (8.8 mg). The L-methionine-supplemented culture experiments with Aspergillus sp. XS-20090B15 were carried out in four groups. Each was cultivated in 5 L of potato glucose liquid medium at 20 °C without shaking for 1 week. Then sterile L-methionine solutions were provided to three groups, and the final concentrations of L-methionine in each group were 2, 1, and 0.5 g/L, respectively. The remaining group, cultivated without additional L-methionine, was used as a blank control. All of the groups were fermented at 20 °C without shaking for 5 weeks. The extraction and purification were performed immediately after harvesting the fungal cultures. Compound 2 was isolated from two of the cultures, together with 1 and 3. The yields of 1, 2, and 3 from the different cultures (each 5 L) were as follows: 36.7 mg (1), 11.6 mg (2), and 2.1 mg (3) from the culture with 2 g/L Lmethionine; 18.4 mg (1), 2.0 mg (2), and 1.9 mg (3) from the culture with 1 g/L L-methionine; 10.2 mg (1), 0 mg (2), and 1.5 mg (3) from the culture with 0.5 g/L L-methionine; 9.9 mg (1), 0 mg (2), and 1.6 mg (3) from the culture without additional L-methionine. Penilumamide B (2): white powder; [α]25D −24 (c 0.15, CH2Cl2); UV (MeOH) λmax (log ε) 220 (2.60), 252 (1.98), 317 (0.76) nm; IR (KBr) νmax 3737, 2364, 1679, 1583, 1504, 1452, 1264, 753 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 1; HRESIMS m/z 501.1544 [M + H]+ (calcd for C22H25N6O6S, 501.1551). Penilumamide C (3): white powder; [α]25D +68 (c 0.50, MeOH); UV (MeOH) λmax (log ε) 222 (2.10), 251 (1.88), 318 (0.56) nm; IR (KBr) νmax 3425, 2950, 2356, 1678, 1586, 1506, 1452, 1292, 1021, 752 cm−1; 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table 1; HRESIMS m/z 555.1256 [M + Na]+ (calcd for C22H24N6O8SNa, 555.1269). Penilumamide D (4): white powder; [α]25D +59 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 217 (2.16), 251 (1.64), 336 (0.50) nm; IR (KBr) νmax 3379, 2944, 2832, 2513, 1455, 1117, 1030, 694 cm−1; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz), see Table 1; HRESIMS m/z 448.1346 [M + Na]+ (calcd for C19H19N7O5Na, 448.1340).

■

ASSOCIATED CONTENT

S Supporting Information *

NMR and MS spectra of 2−5, HPLC profiles of FDAA derivatives of acidic hydrolysates of 2, 4, and 5, and HPLC profiles of three groups of L-methionine-supplemented cultures and the normal culture. These materials are available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*(C.-Y. Wang) Tel/Fax: 86-532-82031536. E-mail: changyun@ ouc.edu.cn. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Nos. 41130858; 41322037; 41176121; 81172977) and the Program for New Century Excellent Talents in University, Ministry of Education of China (No. NCET-11-0472). E

dx.doi.org/10.1021/np5001686 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

■

Article

REFERENCES

(1) (a) Molinski, T. F.; Reynolds, K. A.; Morinaka, B. I. J. Nat. Prod. 2012, 75, 425−431. (b) Wyche, T. P.; Hou, Y.; Braun, D.; Cohen, H. C.; Xiong, M. P.; Bugni, T. S. J. Org. Chem. 2011, 76, 6542−6547. (c) Lin, Z.; Reilly, C. A.; Antemano, R.; Hughen, R. W.; Marett, L.; Concepcion, G. P.; Haygood, M. G.; Olivera, B. M.; Light, A.; Schmidt, E. W. J. Med. Chem. 2011, 54, 3746−3755. (d) Sasaki, H.; Teruya, T.; Fukazawa, H.; Suenaga, K. Tetrahedron 2011, 67, 990− 994. (2) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munroa, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2013, 30, 237−323. (3) Meyer, S. W.; Mordhorst, T. F.; Lee, C.; Jensen, P. R.; Fenical, W.; Kock, M. Org. Biomol. Chem. 2010, 8, 2158−2163. Note: penilumamide is regarded as penilumamide A in the present paper. (4) (a) Cardellina, J. H., II; Meinwald, J. J. Org. Chem. 1981, 46, 4782−4784. (b) Tanino, H.; Takakura, H.; Kakoi, H.; Okada, K.; Inoue, S. Heterocycles 1996, 42, 125−128. (c) Voerman, G.; Cavalli, S.; van der Marel, G. A.; Pfleiderer, W.; van Boom, J. H.; Filippov, D. V. J. Nat. Prod. 2005, 68, 938−941. (5) (a) Chen, M.; Shao, C. L.; Fu, X. M.; Xu, R. F.; Zheng, J. J.; Zhao, D. L.; She, Z. G.; Wang, C. Y. J. Nat. Prod. 2013, 76, 547−553. (b) Qi, J.; Shao, C. L.; Li, Z. Y.; Gan, L. S.; Fu, X. M.; Bian, W. T.; Zhao, H. Y.; Wang, C. Y. J. Nat. Prod. 2013, 76, 571−579. (c) Shao, C. L.; Xu, R. F.; Wei, M. Y.; She, Z. G.; Wang, C. Y. J. Nat. Prod. 2013, 76, 779−782. (d) Yang, K. L.; Wei, M. Y.; Shao, C. L.; Fu, X. M.; Guo, Z. Y.; Xu, R. F.; Zheng, C. J.; She, Z. G.; Lin, Y. C.; Wang, C. Y. J. Nat. Prod. 2012, 75, 935−941. (e) Shao, C. L.; Wu, H. X.; Wang, C. Y.; Liu, Q. A.; Xu, Y.; Wei, M. Y.; Qian, P. Y.; Gu, Y. C.; Zheng, C. J.; She, Z. G.; Lin, Y. C. J. Nat. Prod. 2011, 74, 629−633. (6) (a) Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Nagle, D. G.; Paul, V. J. J. Nat. Prod. 1999, 62, 655−658. (b) Nogle, L. M.; Williamson, R. T.; Gerwick, W. H. J. Nat. Prod. 2001, 64, 716− 719. (c) Baumann, H. I.; Keller, S.; Wolter, F. E.; Nicholson, G. J.; Jung, G.; Süssmuth, R. D.; Jüttner, F. J. Nat. Prod. 2007, 70, 1611− 1615. (d) Gunasekera, S. P.; Ritson-Williams, R.; Paul, V. J. J. Nat. Prod. 2008, 71, 2060−2063. (7) Siemion, I. Z.; Wieland, T.; Pook, K. H. Angew. Chem., Int. Ed. Engl. 1975, 14, 702−703. (8) Marfey, P.; Ottesen, M. Carl. Res. Commun. 1984, 49, 585−590. (9) Pierce, C. G.; Uppuluri, P.; Teistan, A. R.; Wormley, J. F. L.; Mowat, E.; Ramage, G.; Lopez-ribot, J. L. Nat. Protoc. 2008, 3, 1494− 1500. (10) Mosmann, T. J. Immunol. Meth. 1983, 65, 55−63. (11) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112. (12) (a) Kujumgiev, A.; Tsvetkova, I.; Serkedjieva, Y.; Bankova, V.; Christov, R.; Popov, S. J. Ethnopharmacol. 1999, 64, 235−240. (b) Li, Y. L.; But, P. P. H.; Ooi, V. E. C. AntiViral Res. 2005, 68, 1−9. (13) Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Featherstone, R. M. Biochem. Pharmacol. 1961, 7, 88−95.

F

dx.doi.org/10.1021/np5001686 | J. Nat. Prod. XXXX, XXX, XXX−XXX