Psychrophilins E–H and Versicotide C, Cyclic Peptides from the

Sep 23, 2014 - Fax: 0086-532-82033054. ... Four new cyclic peptides, psychrophilins E–H (1–4), possessing a rare amide ... Synthesis of Psychrophi...
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Psychrophilins E−H and Versicotide C, Cyclic Peptides from the Marine-Derived Fungus Aspergillus versicolor ZLN-60 Jixing Peng,† Huquan Gao,† Xiaomin Zhang,† Shuai Wang,‡ Chongming Wu,‡ Qianqun Gu,† Peng Guo,‡ Tianjiao Zhu,*,† and Dehai Li*,† †

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, People’s Republic of China ‡ Pharmacology and Toxicology Research Center, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 100193, People’s Republic of China S Supporting Information *

ABSTRACT: Four new cyclic peptides, psychrophilins E−H (1−4), possessing a rare amide linkage between the carboxylic acid in anthranilic acid (ATA) and the nitrogen from an indole moiety, along with a new ATA-containing hexapeptide, versicotide C (5), were obtained from the culture of the marine-derived fungus Aspergillus versicolor ZLN-60. The structures, including absolute configurations, were elucidated by a combination of HRESIMS, NMR, X-ray crystallography, TDDFT ECD calculations, and Marfey’s method. Versicotide C (5) is the first natural cyclic hexapeptide containing two anthranilic acids. Compounds 1−5 were not cytotoxic, and compound 3 showed potent lipid-lowering effects.

T

he OSMAC (one strain many compounds) approach is an effective way to expand the structural diversity of metabolites of a single fungal strain by systematic alteration of its cultivation parameters including media composition, pH, temperature, oxygen supply, quality and quantity of light, bioreactor platform, and the addition of precursors or enzyme inhibitors.1−3 Our laboratory has successfully applied these methods to maximize the production of new secondary metabolites from marine-derived fungi. Examples of these were cytochalasins from Spicaria elegans KLA034 and melegarin alkaloids and nitrogen-containing sorbicillinoids from Penicillium sp. F23-2.5 In our preliminary screening using the OSMAC strategy, the fungus Aspergillus versicolor ZLN-60, which produces anthranilic acid (ATA)-containing pentapeptides (versicotides A, B)6 and prenylated diphenyl ethers (diorcinols B−E),7 was selected for further study. The HPLC-UV profile of the culture changed dramatically upon alteration of the original liquid medium6 to a rice-based solid medium. Further fractionation of the extract of the solid-phase culture led to the discovery of five new cyclic peptides, psychrophilins E−H (1−4) and versicotide C (5). Herein, we report the details of the isolation, structure elucidation, and biological activities of these new cyclic peptides.

CHCl3−MeOH to give three fractions. These were further fractionated by Sephadex LH-20 column chromatography and semipreparative HPLC to give compounds 1−5. Psychrophilin E (1) was obtained as colorless crystals. Its molecular formula of C25H24N4O4 was established on the basis of the HRESIMS ion at m/z 445.1870 [M + H]+, requiring 12 degrees of unsaturation. Analysis of the 1H NMR and 13C NMR data (Tables 1 and 2) revealed the presence of 24 proton and 25 carbon resonances including four amide carbonyls (δC 166.6, 168.2, 169.9, 171.7), two sp 3 methines, four sp3 methylenes, one methyl, two exchangeable protons, and 14 aromatic carbons including two ortho-substituted benzenes with one of them in an indole moiety, suggesting a peptide with an anthranilic acid and a tryptophan-derived unit, which were

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RESULTS AND DISCUSSION The EtOAc extract (6 g) of the 30-day static fermentation in rice medium was subjected to vacuum liquid chromatography over silica gel using a gradient elution with petroleum ether− © 2014 American Chemical Society and American Society of Pharmacognosy

Received: June 9, 2014 Published: September 23, 2014 2218

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Table 1. 1H NMR Spectroscopic Data (600 MHz, DMSO-d6) for Compounds 1−4 position 2 3 3-OH 6 7 8 9 11 14 15 16 17 18-NH 20 21 22 23 25 26 a

1

2

4.65, m 3.16, m; 2.87 (dd, 12.4, 4.5) 2.88, m

5.35, dd (12.5, 4.5) 3.26, t (12.4); 3.02 (dd, 12.4, 4.5) 3.02, dd (12.1, 4.5)

7.74, 7.35, 7.39, 8.46, 6.77, 7.72, 7.39, 7.65, 7.71, 9.36, 4.53, 1.82, 1.45, 3.37, 1.87, 8.55,

ma ddd (7.7, 7.7, ddd (7.7, 7.7, br d (7.7) s ma ddd (7.7, 7.7, ddd (7.7, 7.7, ma s dd (8.3, 3.9) m; 1.62, m m; 1.25, m m; 2.02, m s d (6.1)

7.67, 7.36, 7.41, 8.49, 6.82, 7.72, 7.40, 7.64, 7.83, 9.30, 4.50, 1.83, 1.43, 3.51, 2.08, 3.18,

1.0) 1.3)

0.9) 1.0)

3

br d (6.8) ma ma d (6.8) s br d (7.6) ma ddd (7.6, 7.6, 1.0) br d (7.6) s dd (8.0, 4.4) m; 1.67, m m; 1.35, m m; 1.93, m s s

4

5.44, d (10.1) 5.18, d (10.1)

5.37, d (10.1) 5.25, d (10.1)

5.94, 7.93, 7.34, 7.39, 8.47, 6.86, 7.65, 7.65, 7.40, 7.73, 9.32, 4.49, 1.81, 1.40, 3.49, 2.08, 3.27,

not det.

br s br d (7.7) ddd (7.7, 7.7, ddd (7.7, 7.7, br d (7.7) s br d (7.6) ddd (7.6, 7.6, ddd (7.6, 7.6, br d (7.6) s dd (8.3, 3.8) m; 1.60, m m; 1.28, m m; 1.90, m s s

1.0) 1.2)

0.8) 1.1)

6.74, 7.25, 7.98, 6.84, 7.72, 7.40, 7.65, 7.60, 9.40, 4.52, 1.83, 1.44, 3.51, 2.08, 3.28,

d (8.2) t (8.2) d (8.2) s br d (7.7) ddd (7.7, 7.7, 0.8) ddd (7.7, 7.7, 0.9) br d (7.7) s dd (7.7, 3.2) m; 1.59 m m; 1.26 m m; 2.12 m s s

Overlapped signals.

Table 2. 13C NMR Spectroscopic Data (150 or 100 MHz, DMSO-d6) for Compounds 1−4 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 a

1a 171.7, 51.8, 28.0, 116.4, 130.2, 119.1, 124.3, 126.0, 116.8, 135.4, 125.4, 166.6, 127.4, 132.1, 126.0, 132.9, 123.5, 134.3, 168.2, 59.3, 24.8, 25.9, 46.7, 169.9, 22.5,

C CH CH2 C C CH CH CH CH C CH C C CH CH CH CH C C CH CH2 CH2 CH2 C CH3

2a 170.3, 52.7, 25.8, 115.9, 129.8, 118.4, 123.8, 125.0, 116.4, 135.0, 125.6, 166.1, 126.5, 131.6, 125.2, 132.3, 122.6, 134.0, 168.0, 59.2, 24.2, 25.5, 46.0, 170.8, 22.1, 31.7,

C CH CH2 C C CH CH CH CH C CH C C CH CH CH CH C C CH CH2 CH2 CH2 C C CH3

3a 169.3, C 58.0, CH 66.2, CH 119. 8, C 128.7, C 120.6, CH 123.4, CH 125.3, CH 116.0, CH 135.2, C 124.0, CH 166.1, C 126.7, C 131.4, CH 124.7, CH 132.2, CH 123.0, CH 133.6, C 167.2, C 58.5, CH 24.7, CH2 24.0, CH2 45.9, CH2 171.2, C 22.0, CH3 31.2, CH3

4b 169.4, 59.4, 65.6, 118.5, 117.4, 149.2, 109.6, 126.8, 107.8, 137.0, 123.1, 166.3, 127.2, 131.6, 125.7, 132.5, 123.5, 133.7, 167.3, 58.2, 25.0, 24.2, 46.2, 171.4, 22.2, 31.7,

C CH CH C C C CH CH CH C CH C C CH CH CH CH C C CH CH2 CH2 CH2 C CH3 CH3

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

the known cyclic nitropeptide psychrophilin A, which contains a nitro group at C-2.8 Finally, the planar structure of 1 was confirmed by attaching an acetamide on C-2 according to the COSY correlation between H-2 (δH 4.65, m) and H-26 (δH 8.55, d, J = 6.06 HZ) and HMBC correlations from H-25 (δH 1.87, s) to C-24 (δC 169.9) and from H-26 to C-24 (Figure 1 and Table 1). To determine the absolute configuration of 1, crystallographic quality crystals were successfully grown and analyzed by X-ray diffraction with Cu Kα irradiation (Flack parameter = −0.08), revealing the absolute configurations at C2 and C-20 to be 2S and 20S (Figure 2). HRESIMS and 1D NMR data for psychrophilin F (2) established the molecular formula C26H26N4O4, a 14 amu increase in the molecular weight compared with 1. The 1D NMR data indicated the appearance of an N-Me (δH 3.18, δC 31.7) and was assigned by the HMBC correlations from H3-26 to C-2 and C-24 (Tables 1 and 2 and Figure 1). The configuration of the L-proline in 2 was determined by the advanced Marfey’s method.9 Biogenetically, the absolute configuration of C-2 was proposed to be the same as 1, which was also supported by the similar CD spectra (Figure 3).8 The HRESIMS adduct peaks indicated the molecular formulas for psychrophilins G (3) and H (4) to be C26H26N4O5 and C26H26N4O6, with one and two more oxygen atoms than compound 2, respectively. Their 1H and 13C NMR

Measured at 150 MHz. bMeasured at 100 MHz.

further identified by 2D NMR data (Figure 1). A proline unit was deduced by COSY correlations between H-20 and H2-21, between H2-21 and H2-22, and between H2-22 and H2-23, together with the HMBC correlations from H2-21 to C-19. The amino acid sequence of 1 was established by the HMBC correlations (Figure 1) and the comparison of the NMR data to 2219

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Figure 4. Key COSY and HMBC correlations of 3 and 4.

Figure 2. X-ray crystal structure of compound 1.

data (Tables 1 and 2) revealed that the structures of 3 and 4 closely resembled compound 2, sharing the same cyclic peptide skeleton. The differences were attributed to the appearance of a C-3 hydroxy group in 3 and at C-3 and C-6 in 4, which were supported by the chemical shifts (δH 5.18 and δC 66.2, CH-3 in 3; δH 5.25 δC 65.6, CH-3, and δC 149.2, C-6 in 4), together with COSY and HMBC correlations (Tables 1 and 2 and Figure 4). The configurations at C-20 of psychrophilins G (3) and H (4) were determined as S from a Marfey’s analysis of the Lproline unit. The anti relationships of H-2 and H-3 were suggested by the vicinal coupling constants between them (3JH‑2,H‑3 = 10.1 Hz for both of 3 and 4), leading to two possible absolute configurations, (2S, 3R, 20S) and (2R, 3S, 20S), for compounds 3 and 4. The NOEs of H-6/H-2 and H-26/H-3/H11 in 3 supported the 2S/3R configurations (Figure 5). The TDDFT-ECD spectra for the two epimers (2S,3R,20S)-3 and (2R,3S,20S)-3 were calculated (Figure 6), and the absolute configuration of 3 was unambiguously determined as (2S, 3R, 20S), because the calculated curves for (2S,3R,20S)-3 agreed with the experimental one. The absolute configuration of compound 4 was also inferred to be the same as 3 based on the similarity of its experimental ECD spectrum (Figure 3).

Figure 5. Selected NOE correlations of 3 and NOESY correlations of 4.

The molecular formula of C28H34N6O6 for versicotide C (5) was provided by its HRESIMS and also supported by NMR data (Table 3), indicating 15 degrees of unsaturation. The 1H and 13C NMR spectra indicated the presence of four NH doublets (δH 12.33, 11.89, 9.13, and 8.28), four characteristic αCH groups (δH/δC 5.19/45.2, 5.19/44.8, 4.57/54.9, and 4.01/ 61.3), and six amide carbonyl signals (δC 171.5, 170.5, 169.7, 169.2, 166.9, and 166.8). Analysis of the 1D and 2D NMR data (Table 3 and Figure 7) of 5 established six amino acid residues: two alanines, two Nmethylalanines, and two ATA residues. The sequence of the six

Figure 3. Experimental ECD spectra of compounds 1, 2, 3, and 4. 2220

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Figure 7. Key COSY and HMBC correlations of 5. Figure 6. B3LYP/6-31+G(d)-calculated ECD spectra of (2S, 3R,20S)3 (red) and (2R,3S,20S)-3 (blue) and the experimental ECD spectrum of 3 (black) (σ = 0.40 eV).

planar structure of versicotide C (5) was thus elucidated as a cyclo-[(Me-Ala)-Ala-ATA-(Me-Ala)-Ala-ATA]. Similar to the previously isolated versicotides A and B,6 compound 5 was also composed of ATA, Ala, and Me-Ala units. The amino acid residues in versicotide C (5) were identified as L-Ala and N-Me-L-Ala by the advanced Marfey’s method,9 applied to the acid hydrolysate of 5. Although the planar structure of 5 is symmetrical, the distinguishing NMR signals of the two N-Me (Table 3) indicated the different configurations of the two corresponding amide bonds considering that they are both formed from NMe-L-Ala. Consistent with previous reports,10 the N-methyl resonance of the cis conformer (syn to carbonyl group) was upfield relative to the trans due to the shielding effect of the carbonyl group, whereby the 4-NMe (δH 2.91, δC 29.6) was deduced as cis, while 17-NMe (δH 3.35, δC 37.6) was trans. The cis/trans conformation was further confirmed by a variabletemperature 1H NMR experiment, which showed a significant decrease in the number of 1H NMR resonances measured at 80 °C compared to that measured at 25 °C (Figure S40, Supporting Information), based upon the rotation of the C− N amide bond at higher temperature. The new cyclic peptides 1−5 were evaluated for cytotoxicities against A-549, HeLa, and SMMC-7721 cancer cells with the sulforhodamine B (SRB) method,11 but none of them were toxic (IC50 >100 μM). In a test for lipid-lowering activity in HepG2 hepatocarcinoma cells, compound 3 exhibited potent lipid-lowering effects at a dose of 10 μM as assessed by Oil Red O staining (Figure 8).12 In summary, five new cyclic peptides were disclosed from A. versicolor ZLN-60 guided by the OSMAC approach, indicating that the Aspergillus genus is a pliable source of secondary metabolites with high structural diversity and interesting bioactivities.13 Psychrophilins represent rare fungal cyclic peptides containing amide groups composed of anthranilic acid and indole motifs in the macrocycle. Only four prior examples of such were reported to date (psychrophilins A−D from Penicillium sp.).8 Compounds 1−4 extend the psychrophilin class from four compounds to the present eight. It is worth noting that all of the compounds isolated contain one or two anthranilic acid residues, which are relatively rare in cyclic peptides, and versicotide C (5) is the first natural cyclic hexapeptide containing two anthranilic acids. Biogenetically, anthranilic acid can be produced by two possible pathways, the shikimic acid pathway, as an intermediate during the synthesis of tryptophan, and the kynurenine pathway, in which tryptophan is first metabolized to kynurenine and subsequently to ATA by kynureninase.14 Further studies are needed to

Table 3. NMR Spectroscopic Data (400 MHz, DMSO-d6) for Compound 5 position 1 1-NH 2 3 4 4-NMe 5 6 7 7-NH 8 9 10 11 12 13 14 14-NH 15 16 17 17-NMe 18 19 20 20-NH 21 22 23 24 25 26

δC, type

δH (J in Hz)

169.7, C 54.9, 16.8, 170.5, 29.6, 44.8, 17.6, 166.8,

CH CH3 C CH3 CH CH3 C

118.3, 127.7, 121.7, 132.4, 118.4, 139.9, 169.2,

C CH CH CH CH C C

61.3, 12.3, 171.5, 37.6, 45.2, 17.1, 166.9,

CH CH3 C CH3 CH CH3 C

115.5, 128.2, 121.4, 132.2, 118.3, 140.1,

C CH CH CH CH C

12.33, s 4.57, q (7.0) 1.56, d (7.0) 2.91, s 5.19, m 1.22, d (6.5) 9.13, d (9.1) 7.92, 7.01, 7.43, 8.60,

d (8.4) br t (8.4) br t (8.4) d (8.4)

11.89, s 4.01, q (6.8) 1.37, d (6.8) 3.35, s 5.19, m 1.33, d (7.1) 8.28, d (7.3) 7.91, 6.85, 7.01, 7.91,

d (7.6) br t (7.6) br t (7.6) d (7.6)

amino acid units was confirmed on the basis of the observed HMBC correlations from alpha and N-methyl protons, as well as amide NH signals to the carbonyl carbons including 1-NH (ATA6) with the carbonyl carbon C-1 (Me-Ala1), H-2 and Nmethyl protons (4-NMe, Me-Ala1) with amide carbon C-4 (Ala2), H-5 (Ala2) with carbonyl carbon C-7 (ATA3), 14-NH (ATA3) with amide carbon C-14 (Me-Ala4), N-methyl protons (17-NMe, Me-Ala4) with carbonyl carbon C-17 (Ala5), and H18 (Ala5) with carbonyl carbon C-20 (ATA6) (Figure 7). The 2221

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Psychrophilin D (1): colorless crystals; mp 190 °C; [α]25D +96 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.2), 244 (4.0), 302 (3.7); ECD (2.25 × 10−3 M, MeOH) λmax (Δε) 374 (+0.06), 316 (−1.26), 288 (+2.81), 273.5 (+2.24), 256.5sh (+0.20), 233 (−12.97), 225 (−11.82), 220 (−12.76) nm; IR (KBr) νmax 3375, 1699, 1636, 1449, 1363 cm−1; 1H NMR and 13C NMR (see Tables 1 and 2); HRESIMS [M + H]+ m/z 445.1875 (calcd for C25H25N4O4, 445.1870). Psychrophilin E (2): white, amorphous powder; [α]25D +120 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.3), 244 (4.1), 302 (3.7); ECD (2.18 × 10−3 M, MeOH) λmax (Δε) 371 (+0.01), 320 (−1.01), 290.5 (+2.63), 273 (+5.30), 255sh (+0.10), 232.8 (−11.62) nm; IR (KBr) νmax 3372, 1699, 1637, 1449, 1361 cm−1; 1H NMR and 13C NMR (see Tables 1 and 2); HRESIMS [M + H]+ m/z 459.2027 (calcd for C26H27N4O4, 459.2027). Psychrophilin F (3): white, amorphous powder; [α]25D +29 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.4), 244 (4.2), 304 (3.7); ECD (2.10 × 10−3 M, MeOH) λmax (Δε) 367 (−0.07), 314 (−0.76), 290.5 (+1.76), 255.8sh (+0.18), 223 (−10.75) nm; IR (KBr) νmax 3300, 1696, 1630, 1523, 1449, 1268, 1169, 1088, 754 cm−1; 1H NMR and 13C NMR (see Tables 1 and 2); HRESIMS [M + H]+ m/z 475.1980 (calcd for C26H27N4O5, 475.1976). Psychrophilin G (4): white, amorphous powder; [α]25D +31 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (4.3), 244 (4.1), 304 (3.7); ECD (1.02 × 10−3 M, MeOH) λmax (Δε) 383 (+0.26), 329 (−0.71), 297 (+2.25), 257.3sh (−0.07), 236.5 (−8.00), 228.3 (−3.78), 222.2 (−6.92) nm; IR (KBr) νmax 3303, 1696, 1631, 1525, 1449, 1268, 1169, 1086 cm−1; 1H NMR and 13C NMR (see Tables 1 and 2); HRESIMS [M + H]+ m/z 491.1930 (calcd for C26H27N4O6, 491.1925). Versicotide C (5): white, amorphous powder; [α]25D +100 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 216 (0.1), 290 (2.6); IR (KBr) νmax 3356, 3190, 2906, 1664, 1613, 1426, 756 cm−1; 1H NMR and 13C NMR (see Table 3); HRESIMS [M + H]+ m/z 551.2616 (calcd for C28H35N6O6, 551.2613). X-ray Crystallographic Analysis of Compound 1. Singlecrystal X-ray diffraction data were collected on an Agilent Gemini Ultra diffractometer with Cu Kα radiation (λ = 1.541 84 Å). The structure was solved by direct methods (SHELXS-97) and refined using full-matrix least-squares difference Fourier techniques. Carbon, oxygen, and nitrogen atoms were refined anisotropically. Hydrogen atoms were either refined freely with isotropic displacement parameters or positioned with an idealized geometry and refined riding on their parent C atoms. Crystals suitable for X-ray diffraction were obtained by slow evaporation of a solution in MeOH−H2O. Crystallographic data (excluding structure factors) for 1 have been deposited with the Cambridge Crystallographic Data Centre: CCDC reference number 973003. These data can be obtained, free of charge, from the Cambridge Crystallographic Data Centre via http://www. ccdc.cam.ac.uk/data_request/cif. Crystal data for 1: orthorhombic, C25H24N4O4, space group P212121 with a = 12.396 10(10) Å, b = 12.968 80(10) Å, c = 13.795 70(10) Å, α = 90°, β = 90°, γ = 90°, V = 2217.83(3) Å3, Z = 4, T = 292(2) K, Dc = 1.331 mg/mm3, μ = 0.752 mm−1, and F (000) = 936. Crystal size: 0.32 × 0.3 × 0.24 mm3. Independent reflections: 4129 with Rint = 0.0218. The final agreement factors are R1 = 0.0272 and wR2 = 0.0768 [I > 2σ(I)]. Flack parameter = −0.08(15). Absolute Configuration of Amino Acids. The acid hydrolysates of 2−5 were redissolved in H2O (50 μL) separately, and then 0.25 μM L-FDAA in 100 μL of acetone was added, followed by 1 N NaHCO3 (25 μL). The mixtures were heated for 1 h at 43 °C. After cooling to room temperature, the reaction was quenched by the addition 2 N HCl (25 μL). Finally the resulting solution was filtered through a small 4.5 μm filter and stored in the freezer until HPLC analysis. Amino acid standards were derivatized with L-FDAA in a similar manner. The resulting L-FDAA derivatives of compounds 2−4, L- and D-alanine, and L- and D-Me-alanine were separately analyzed by reversed-phase HPLC (5 × 250 mm YMC C18 column, 5 μm, with a linear gradient of MeCN (A) and 0.05% aqueous TFA (B) from 5% to 55% A over 55 min at a flow rate of 1 mL/min, UV detection at 320 nm). The resulting LFDAA derivatives of 5, L- and D-proline, were analyzed by reversed-

Figure 8. Effects of compounds on oleic acid-elicited intracellular lipid accumulation. Positive control: simvastatin; blank: DMEM; OA: oleic acid. Neutral lipids were determined by spectrophotometry at 358 nm after Oil Red O staining. Bars depict the means ± SEM in triplicate. ## p < 0.001, OA vs blank; **p < 0.01, test group vs OA group.

determine which of these routes is responsible for the biosynthesis of ATA in A. versicolor.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. CD spectra were measured on a JASCO J-715 spectropolarimeter. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer on KBr discs. NMR spectra were recorded on JEOL JNMECP 600 and Bruker-400 spectrometers using TMS as an internal standard. Ectothermic NMR spectra were recorded on Varian-500 spectrometers. HRESIMS spectra were measured on a Micromass EI-4000 (Autospec-UltimaTOF). X-ray crystal data were measured on an Agilent Gemini Ultra diffractometer (Cu Kα radiation). Semipreparative HPLC was performed using an ODS column (YMC-Pack ODS-A, 5 μm, 10 × 250 mm, 4 mL/min). TLC and column chromatography (CC) were performed on plates precoated with silica gel GF254 (10−40 μm) and over silica gel (200−300 mesh, Qingdao Marine Chemical Factory), respectively. Size-exclusion chromatography was performed using Sephadex LH-20 (GE Healthcare). The seawater was collected from Huiquan Bay, Yellow Sea, China. Fungal Material. The fungal strain was isolated and identified as previously described.6 Fermentation and Extraction. The strain was cultured under static conditions at about 28 °C in 1 L Erlenmeyer flasks containing rice medium (rice 80 g, seawater 120 mL). After 30 days, the cultures of 100 flasks were extracted with MeOH. The MeOH extract was evaporated under reduced pressure to afford an aqueous solution and then extracted with EtOAc. The EtOAc solution was concentrated under reduced pressure to give an organic extract (6.0 g). Purification. The organic extract was subjected to vacuum liquid chromatography over a silica gel column using a gradient elution with petroleum ether−CHCl3−MeOH to give three fractions. Fraction 1 was subjected to Sephadex LH-20 column chromatography eluting with MeOH and then purified by semipreparative HPLC (55:45 MeOH−H2O, 4 mL/min) to give compound 1 (8 mg, tR 13 min), compound 2 (22 mg, tR 18 min), and compound 3 (30 mg, tR 10 min). Fraction 2 was separated by Sephadex LH-20 eluting with CHCl3− MeOH (1:1) and then by semipreparative HPLC (55:45 MeOH− H2O, 4 mL/min) to afford compound 4 (6 mg, tR 15 min) and compound 5 (60 mg, tR 19 min). 2222

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phase HPLC (5 × 250 mm YMC C18 column, 5 μm), with a linear gradient of MeCN (A) and 0.05% aqueous TFA (B) from 10% to 50% A over 60 min at a flow rate of 1 mL/min, UV detection at 320 nm. Each chromatographic peak was identified by comparing its retention times for the L-FDAA derivatives of the L- and D-amino acid standards. The standards gave the following retention times (in min): 40.20 for LFDAA, 38.61 for L-Ala, 43.92 for D-Ala, 41.15 for L-Me-Ala, 39.63 for D-Me-Ala, 37.51 for L-Pro, 38.73 for D-Pro. The analysis gave retention times (in min) of 35.7, 38.61, and 41.15, establishing the S configuration for all the amino acid residues. Computation Section. Conformational searches were run employing the “systematic” procedure implemented in Spartan’14,15 using MMFF (Merck molecular force field). All MMFF minima were reoptimized with DFT calculations at the B3LYP/6-31+G(d) level using the Gaussian 09 program.16 The geometry was optimized starting from various initial conformations, with vibrational frequency calculations confirming the presence of minima. Time-dependent DFT calculations were performed on the lowest energy conformations (>5% population) for each configuration using 20 excited states and using a polarizable continuum model (CPCM) for MeOH. ECD spectra were generated using the program SpecDis17 by applying a Gaussian band shape with 0.40 eV width, from dipole-length rotational strengths. The dipole velocity forms yielded negligible differences. The spectra of the conformers were combined using Boltzmann weighting, with the lowest energy conformations accounting for about 97% of the weights. The calculated spectrum was blue-shifted by 25 nm to facilitate comparison to the experimental data. Acid Hydrolysis of Peptides. Compounds 2−5 (1 mg each) were separately dissolved in degassed 6 N HCl (1 mL) and heated at 105 °C for 14 h. After cooling, the solvent was removed in vacuo. Cell-Based Lipid Accumulation Assay. Before the bioassay, the compounds were tested for toxic activity toward HepG2 cells, showing no toxicity up to 100 μM. HepG2 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum and penicillin/ streptomycin (100 μg/mL). The cells with 70−80% confluence were incubated in DMEM + oleic acid (100 μM) for 12 h, then were treated with the compounds (each, 10 μM) and the positive control simvastatin in DMEM/100 μM oleic acid with DMEM/100 μM oleic acid as a blank for an additional 6 h. Subsequently, the cells were subjected to Oil Red O staining determination as described previously.12 Each experiment (n = 8 for Oil Red O staining or n = 3 for TC and TG determination) was repeated in triplicate.

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ASSOCIATED CONTENT

S Supporting Information *

Computational data, HPLC analysis of the fungal metabolite under different culture conditions, Marfey’s experimental data of 2−5, as well as NMR spectra for compounds 1−5. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(T. Zhu) Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail: [email protected]. *(D. Li) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Nos. 41176120 and 21372208), the NSFC-Shandong Joint Fund (No. U1406402), the National High Technology Research and Development Program of China (No. 2013AA092901), and the Program for New Century Excellent Talents in University (No. NCET-12-0499). 2223

dx.doi.org/10.1021/np500469b | J. Nat. Prod. 2014, 77, 2218−2223