Antifungal and Antiviral Cyclic Peptides from the Deep-Sea-Derived

Article pubs.acs.org/JAFC

Antifungal and Antiviral Cyclic Peptides from the Deep-Sea-Derived Fungus Simplicillium obclavatum EIODSF 020 Xiao Liang,†,‡ Xu-Hua Nong,† Zhong-Hui Huang,†,‡ and Shu-Hua Qi*,† †

Key Laboratory of Tropical Marine Bio-resources and Ecology/Guangdong Key Laboratory of Marine Materia Medica/RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou, 510301 Guangdong, China ‡ University of Chinese Academy of Sciences, Beijing 100049, China S Supporting Information *

ABSTRACT: A new linear peptide simplicilliumtide I (1) and four new cyclic peptides simplicilliumtides J−M (2−5) together with known analogues verlamelins A and B (6 and 7) were isolated from the deep-sea-derived fungal strain Simplicillium obclavatum EIODSF 020. Their structures were elucidated by spectroscopic analysis, and their absolute configurations were further confirmed by chemical structural modification, Marfey’s and Mosher’s methods. Compounds 2, 6, and 7 showed significant antifungal activity toward Aspergillus versicolor and Curvularia australiensis and also had obvious antiviral activity toward HSV-1 with IC50 values of 14.0, 16.7, and 15.6 μM, respectively. The structure−bioactivity relationship of this type of cyclic peptide was also discussed. This is the first time to discuss the effects of the lactone linkage and the substituent group of the fatty acid chain fragment on the bioactivity of this type of cyclic peptides. This is also the first time to report the antiviral activity of these cyclic peptides. KEYWORDS: deep-sea-derived fungus, Simplicillium obclavatum, peptide, antifungal, antiviral, structure−bioactivity relationship

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INTRODUCTION

Fungi are important sources of novel bioactive compounds that are considered to be interesting synthetic models or important new lead compounds for medicine as well as for plant protection.1−5 Fungus Simplicillium lamellicola, previously known as Acremonium strictum BCP, was lately given the name based on 28S rRNA gene and ITS regions. A. strictum BCP could produce cyclic hexapeptide verlamelin6 and was used as a microbial fungicide with the mechanism of mycoparasitism and antibiosis.7−9 In our previous study, we had found eight linear peptides (namely, simplicilliumtides A− H) from a deep-sea-derived fungal strain Simplicillium obclavatum EIODSF 020.10 Now, in our continued study, a new linear peptide simplicilliumtide I (1) and four new cyclic hexapeptides simplicilliumtides J−M (2−5) together with known analogues verlamelins A and B (6 and 7)11,12 were further isolated from the same crude extract of the fungal strain (Figure 1). Verlamelin A is a cyclic hexadepsipeptide antibiotic originally isolated from Verticillium lamellicola in 198011 with antifungal activity toward phytopathogenic fungi in vitro and in vivo;13,14 then it was also isolated from an entomopathogenic fungus Lecanicillium sp. HF627 in 2014 together with its analogue verlamelin B, and its absolute configuration was determined for the first time.12 At the same time a gene cluster responsible for the biosynthesis of verlamelin was also identified.15 In this article, we report the isolation, structure elucidation, and antifungal and antiviral activities of the peptides (1−7). Their structure−bioactivity relationship is also discussed. © 2017 American Chemical Society

Figure 1. Structures of compounds 1−7.

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MATERIALS AND METHODS

General Experimental Procedures. The procedures were the same as previously reported.10 Details are described in the Supporting Information.

Received: Revised: Accepted: Published: 5114

March 20, 2017 June 1, 2017 June 4, 2017 June 5, 2017 DOI: 10.1021/acs.jafc.7b01238 J. Agric. Food Chem. 2017, 65, 5114−5121

Article

Journal of Agricultural and Food Chemistry Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data for Compound 1 (in DMSO-d6, δ ppm)a compound 1 position ABA 1 2 3 4 5 6 7 NH L-Met(O) 1 2 3 4 5

δC, type 169.5, C 117.2, C 140.2,C 119.8, CH/119.9, CH 133.8, CH 122.9, CH 131.1, CH

170.0, C 53.7, CH 23.7, CH2/23.9, CH2 49.2, CH2/49.3, CH2 37.7, CH3

NH a

δH (J in Hz)

position

8.50 (d, 8.0)/8.51 (d, 8.5) 7.60 (t, 7.5) 7.17 (m) 8.00 (d, 8.0) 11.68 (br s)

4.49 2.09 2.74 2.56

(m) (m)/2.31 (m) (m)/2.85 (m) (s)

8.63 (d, 7.5)

D-N-Me-Phe 1 2 3 4 5, 9 6, 8 7 N−CH3 L-allo-Ile 1 2 3 4 5 6 NH2

δC, type 169.7, C 57.5, CH 34.1, CH2 137.3, C 128.7, CH 128.2, CH 126.4, CH 31.3, CH3 169.6, C 53.4, CH 34.8, CH 12.3, CH3 25.0, CH2 11.5, CH3

δH (J in Hz)

5.54 (dd, 5.0, 10.0) 3.37 (dd, 3.5, 14.5) 3.06 (m) 7.33 7.27 7.20 2.98

(d, 7.5) (t, 7.5) (m) (s)

4.14 1.15 0.29 0.94 0.71 7.89

(br s) (m) (t, 6.0) (m), 1.19 (m) (t, 7.0) (br s)

“/” indicates two sets for some data. (3.25) nm; IR (film) νmax 3414, 3325, 3244, 2963, 1633, 1516, 1448, 1431, 1244, 1196,1182, 1140, 1024 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 682.3172 [M + Na] + (calcd. for C31H45N7O9Na, 682.3171). Determination of Amino Acid Residues’ Absolute Configurations of 1−5 (Marfey’s Method17). The procedures were similar to the previous report.10 Details are described in the Supporting Information. Methylation of 2 and 3. Compounds 2 (11 mg) and 3 (11 mg) were dissolved in 550 μL of dried methanol, respectively. Then 60 μL of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 60 μL of CH3I were added, and the mixture was incubated at 50 °C for 4 h. The reaction was quenched by diluting with water; then it was concentrated, and the residual water was extracted with ethyl acetate. The organic fractions were collected and concentrated in vacuo. The extracts were purified by (SP-RP) HPLC to yield 2a (tR = 33.5 min, 9 mg) and 3a (tR = 48.1 min, 13 mg) eluting with CH3CN/H2O/TFA (v/v/v 72:28:0.03, 5 mL/min) and CH3CN/H2O/TFA (v/v/v 41:59:0.03, 5 mL/min), respectively. Methanolysis Reaction of 2a and 3a. Details were described in the Supporting Information. Briefly, the methyl ethers (2a or 3a) were dissolved in 2 mL of 0.5 M methanol solution of sodium methoxide and stirred at room temperature (20 °C) for 9 h. Then 1 mL of 2 M hydrochloric acid was added to quench the reaction. The mixture was purified by HPLC to yield 2b (2.0 mg) and 3b (2.8 mg), respectively. The recovered samples of 2a or 3a were reused for the reaction as the above-described. Totally, 4.5 mg of 2b and 5.0 mg of 3b were obtained, respectively. Compound 2b. HRESIMS m/z 968.5686 [M + Na]+ (calcd for C48H79N7O12Na, 968.5679). Compound 3b. HRESIMS m/z 968.5315 [M + Na]+ (calcd for C47H75N7O13Na, 968.5315). Preparation of the (R)- and (S)-MTPA Ester Derivatives of 2b and 3b. Details are described in the Supporting Information. Briefly, 2b was divided equally into two portions and dried overnight in vacuo; then 500 μL of deuterated pyridine, 1.0 mg of 4-dimethyl aminopyridine (DMAP), and 20 μL of (R)-(−) or (S)-(+)-methoxyα-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl) were added, respectively. The mixtures were stirred at room temperature and quenched by adding water after 17 h to give the (S)- and (R)-MTPA ester derivatives 2c (1.5 mg) and 2d (1.8 mg), respectively. The (S)and (R)-MTPA ester derivatives of 3b were prepared by the above methods to give 3c (0.7 mg) and 3d (1.0 mg). Their 1H and 1H−1H COSY NMR spectra were detected using CD3OD as solvent.

Fungal Materials. The fungus S. obclavatum EIODSF 020 was isolated from a marine sediment sample collected in the East Indian Ocean and identified by Dr. Xiaoyong Zhang.16 Please see Supporting Information. Fermentation and Extraction. The procedures were the same as previously reported.10 Details are described in the Supporting Information. Briefly, the strain was inoculated in Erlenmeyer flasks containing liquid medium at 28 °C for 2 days as seed cultures, transferred into 300 × 1 L Erlenmeyer flasks, and cultured under static condition at 26 °C for 30 days. Totally, 90 L of fermentation broth was harvested to yield a crude extract (115.7 g). Isolation and Purification. The procedures were similar to the previous report.10 Details are described in the Supporting Information. Briefly, the crude extract was repeatly purifed by silica gel column, ODS column, Sephadex LH-20 column, and high-performance liquid chromatography (HPLC) to offer compounds 1 (10.0 mg), 2 (53.6 mg), 3 (42.2 mg), 4 (5.4 mg), 5 (5.0 mg), 6 (75.7 mg), and 7 (13.2 mg). Simplicilliumtide I (1). Pale yellow oil; [α]25D + 61 (c 0.169, CH3OH); UV (CH3OH) λmax (log ε) 211 (4.20), 254 (3.81), 294 (3.26) nm; IR (film) νmax 3366, 3235, 1647, 1585, 1497, 1448, 1373, 1296, 1254, 1200, 1180, 1134 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 581.2399 [M + Na]+ (calcd. for C28H38N4O6SNa, 581.2404). Simplicilliumtide J (2). Colorless solid; [α]25D − 10 (c 0.176, CH3OH); UV (CH3OH) λmax (log ε) 204 (4.27), 225 (3.85), 278 (2.74) nm; IR (film) νmax 3294, 2959, 2926, 2855, 1732, 1634, 1539, 1516, 1456, 1437, 1244, 1134 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 900.5431 [M + H]+ (calcd. for C46H74N7O11, 900.5441). Simplicilliumtide K (3). Colorless solid, [α]25D − 12 (c 0.119, CH3OH); UV (CH3OH) λmax (log ε) 203 (4.30), 225 (3.87), 277 (2.89) nm; IR (film) νmax 3294, 2932, 2859, 1636, 1539, 1516, 1454, 1373, 1263, 1236, 1202, 1174, 1134, 1026 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 922.4892 [M + Na]+ (calcd. for C45H69N7O12Na, 922.4896). Simplicilliumtide L (4). Colorless solid, [α]25D − 10 (c 0.128, CH3OH); UV (CH3OH) λmax (log ε) 203 (4.29), 225 (3.89), 277 (3.05) nm; IR (film) νmax 3292, 2936, 1645, 1539, 1516, 1456, 1375, 1240, 1202, 1182, 1136, 1026 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 922.4893 [M + Na]+ (calcd. for C45H69N7O12Na, 922.4896). Simplicilliumtide M (5). Colorless solid, [α]25D + 14 (c 0.083, CH3OH); UV (CH3OH) λmax (log ε) 203 (4.37), 225 (3.98), 277 5115

DOI: 10.1021/acs.jafc.7b01238 J. Agric. Food Chem. 2017, 65, 5114−5121

Article

Journal of Agricultural and Food Chemistry Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Data for Compounds 2−5 (in DMSO-d6, δ ppm)a compound position

2 δC, type

δH (J in Hz)

3 δC, type

δH (J in Hz)

4 δC, type

5

δH (J in Hz)

δC, type

δH (J in Hz)

L-Val/L-allo-Ile

1 2 3 4 5 NH

171.2, C 56.5, CH 35.6, CH2 14.8, CH3 25.0, CH2 11.2, CH3

4.14 (dd, 6.0, 7.5) 1.84 (overlapped) 0.84 (d, overlapped) 1.17 (overlapped), 1.11 (overlapped) 0.78 (t, 7.0)

170.4, C 59.3, CH 29.1, CH 18.4, CH3 18.9, CH3

3.85 (t, 7.0) 1.99 (m) 0.83 (t, 6.5) 0.83 (t, 6.5)

170.4, C 59.3, CH 29.1, CH 18.4, CH3 18.9, CH3

7.78 (d, 7.0)

3.84 (t, 7.0) 1.99 (m) 0.83 (t, 6.5) 0.83 (t, 6.5)

172.7, C 59.9, CH 28.6, CH 19.6, CH3 19.4, CH3

8.46 (d, 7.5)

3.91 (m) 1.72 (m) 0.77 (d, 6.5) 0.58 (d, 6.5) 8.19 (d,7.0)

D-Tyr

1 2 3 4 5, 9 6, 8 7 7-OH NH L-Gln 1 2 3 4 5 CONH2

172.9, C 53.9, CH 38.0, CH2 127.4, C 130.2, CH 114.7, CH 155.7, C

3 4 5

7.06 (d, 8.5) 6.61 (d, 8.0)

172.1, C 54.0, CH 38.0, CH2 127.4, C 130.2, CH 114.7, CH 155.7, C

9.12 (s) 7.53 (d, 8.0) 170.8, C 52.6, CH 26.6, CH2 31.8, CH2 173.5, C

NH L-Pro 1 2

4.68 (dd, 9.0, 15.0) 2.74 (m)

170.8, C 60.2, CH 29.1, CH2 24.0, CH2 46.7, CH2

4.04 (m) 1.70 (m) , 1.91 (m) 2.02 (m)

4.64 (dd, 8.5, 14.0) 2.75 (m)

7.05 (d, 8.5) 6.61 (d, 8.5)

9.09 (br s) 7.51 (d, 8.0) 170.9, C 52.6, CH 26.7, CH2 31.8, CH2 173.6, C

4.05 (m) 1.79 (m), 1.91 (m) 2.02 (m)

6.74 (s), 7.22 (s)

6.74 (s), 7.21 (s)

7.79 (overlapped)

7.79 (overlapped)

4.33 (dd, 4.0, 9.0) 2.11 (overlapped), 1.84 (overlapped) 1.83 (overlapped) 3.49 (m), 3.63 (m)

172.1, C 54.0, CH 38.0, CH2 127.4, C 130.2, CH 114.7, CH 155.7, C

170.8, C 60.1, CH 29.1, CH2 24.1, CH2 46.7, CH2

4.33 (dd, 5.0, 9.0) 2.11 (m), 1.84 (m) 1.83 (m) 3.48 (m), 3.62 (m)

4.62 (m) 2.75 (m)

7.04 (d, 8.5) 6.61 (d, 8.5)

170.2, C 54.8, CH 38.0, CH2 127.8, C 130.4, CH 115.3, CH 156.2, C

9.12 (s) 7.52 (d, 8.5) 170.9, C 52.6, CH 26.7, CH2 31.7, CH2 173.6, C

4.05 (m) 1.69 (m), 1.91 (m) 2.02 (m)

29.1, CH2 24.0, CH2 46.7, CH2

6.94 (d, 8.0) 6.61 (d, 8.0)

9.13 (s) 7.29 (d, 6.0) 171.0, C 52.7, CH 26.6, CH2 32.2, CH2 174.2, C

6.74 (s), 7.22 (s) 7.80 (overlapped) 170.8, C 60.1, CH

4.48 (m) 2.75 (m)

4.16 (m) 1.64 (m), 2.16 (m) 2.00 (t, 7.5)

6.72 (s), 7.27 (s) 8.16 (d, 8.5)

172.2, C 61.8, CH

4.16 (m)

3.60 (m), 3.48 (m)

30.1, CH2 24.8, CH2 47.4, CH2

2.17 (m), 1.84 (m) 1.85 (m), 1.90 (m) 3.47 (m), 3.74 (m)

4.48 (m)

171.4, C 47.8, CH

4.57 (m)

4.33 (dd, 4.0, 9.0) 2.11 (m), 1.83 (m) 1.83 (m)

D-Ala

1 2 3

170.9, C 46.8, CH2 16.3, CH3

NH

4.50 (m) 1.18 (d, 6.5)

170.8, C 46.8, CH2 16.2, CH3

7.77 (overlapped)

170.8, C 46.8, CH

4.49 (m) 1.18 (d, 6.5)

16.2, CH3

7.77 (overlapped)

1.18 (d, 6.5)

18.2, CH3

7.78 (overlapped)

1.22 (d, 6.5) 7.44 (d, 6.0)

D-allo-Thr

1 2

170.3, C 58.3, CH

4.21 (dd, 7.0, 8.5)

170.7, C 58.2, CH

4.21 (dd, 7.0, 8.5)

3

66.3, CH

4.00 (m)

66.3, CH

4.00 (m)

4

19.8, CH3

1.07 (d, 6.5)

19.9, CH3

1.08 (d, 6.5)

3-OH NH

5.25 (br s) 7.48 (d, 9.0)

ND 7.48 (d, 9.0)

5116

170.7, C 58.2, CH 66.3, CH2 19.9, CH3

4.20 (dd, 7.0, 8.5) 4.00 (m) 1.08 (d, 6.5) 5.27 (br s) 7.49 (d, 9.0)

169.9, C 59.0, CH

4.19 (m)

66.7, CH

4.06 (m)

18.9, CH3

1.08 (d, 6.0) 4.84 (br s) 8.36 (d, 8.0)

DOI: 10.1021/acs.jafc.7b01238 J. Agric. Food Chem. 2017, 65, 5114−5121

Article

Journal of Agricultural and Food Chemistry Table 2. continued compound position

2 δC, type

δH (J in Hz)

3 δC, type

4

δH (J in Hz)

δC, type

(S)-5-hydroxytetradecanoic acid/(S)-5-hydroxy-13-ketotetradecanoic acid/5-hydroxy-12-ketotetradecanoic acid 1 172.1, C 172.6, C 172.6, C 2 34.7, 2.14 (overlapped), 1.98 34.6, 2.16 (m, overlapped), 1.98 (m, 34.6, CH2 (overlapped) CH2 overlapped) CH2 1.31 (m), 1.36 (m) 19.7, 1.29 (m), 1.36 (m) 19.7, 3 19.9, CH2 CH2 CH2 4 32.6, 1.48 (m), 1.33 (overlapped) 32.5, 1.48 (m), 1.35 (m,overlapped) 32.5, CH2 CH2 CH2 5 73.5, CH 4.82 (m) 73.2, CH 4.80 (m) 73.2, CH 6 33.5, 1.44 (m) 33.4, 1.43 (m) 33.3, CH2 CH2 CH2 7 25.4, 1.23 (overlapped) 24.9, 1.22 (overlapped) 24.8, CH2 CH2 CH2 1.17−1.24 (overlapped) 28.7, 1.15−1.28 (overlapped) 28.5, 8 28.9, CH2 CH2 CH2 9 28.8, 1.17−1.24 (overlapped) 28.5, 1.15−1.28 (overlapped) 28.4, CH2 CH2 CH2 1.17−1.24 (overlapped) 28.4, 1.15−1.28 (overlapped) 23.1, 10 29.0, CH2 CH2 CH2 11 28.6, 1.17−1.24 (overlapped) 23.1, 1.42 (m, overlapped) 41.3, CH2 CH2 CH2 1.22 (overlapped) 42.6, 2.38 (t, 7.5) 210.9,C 12 31.2, CH2 CH2 13 22.0, 1.23 (overlapped) 208.5,C 34.8, CH2 CH2 14 13.9, 0.84 (t, overlapped) 29.6, 2.05 (s) 7.6, CH2 CH3 CH3 a

5

δH (J in Hz)

δC, type

δH (J in Hz)

2.16 (m), 1.97 (m) 1.29 (m), 1.36 (m) 1.47 (m), 1.35 (m) 4.80 (m) 1.42 (m) 2.20 (m, overlapped) 1.14−1.28 (overlapped) 1.14−1.28 (overlapped) 1.41 (m) 2.37 (m)

2.39 (m) 0.90 (t, 7.0)

ND: not detected.

Figure 2. Key 1H−1H COSY, HMBC correlations, and (+)-ESIMS/MS fragments of 1. Compound 2c. HRESIMS m/z 1394.6178 [M + Cl − H2O]− (calcd. for C68H91N7O15ClF6, 1394.6171). Compound 2d. HRESIMS m/z 1394.6195 [M + Cl − H2O]− (calcd. for C68H91N7O15ClF6, 1394.6171). Compound 3c. HRESIMS m/z 1382.6025 [M + Na − H2O]+ (calcd. for C67H87N7O16NaF6, 1382.6006). Compound 3d. HRESIMS m/z 1382.6055 [M + Na − H2O]+ (calcd. for C67H87N7O16NaF6, 1382.6006). Antifungal Assay. The antifungal activities against two marine gorgonian pathogenic fungal strains Aspergillus versicolor SCSGAF 0096 and A. sydowii SCSGAF 0035 (cultured in PDA medium with 3% sea salt), a phytopathogenic fungus Curvularia australiensis (cultured in SDAY medium), and a human pathogenic fungal strain Candida albicans SC5314 (cultured in YPD medium) were tested by disc diffusion method. The tested fungi were cultured on the nutrient agar plate at 30 °C for 3−5 days. Then, the aqueous suspensions of spores for inoculation were prepared with water containing 0.02% (v/v) Tween 80 (106−107 spores/mL). Tested samples were dissolved in DMSO (20 mg/mL). Spore suspensions (100 μL) were added on the nutrient agar plate and evenly spread on it. Paper discs (d = 5 mm) loaded with 50 μg samples were placed on the inoculated plate. Amphotericin B (2.5 μg/disc) and ketoconazole (2.5 μg/disc) were used as positive controls, respectively. The paper disc loaded with 2.5 μL of DMSO was used as the negative control. The inhibition effect was observed after culturing at 30 °C for 2−5 days.

Then the minimum inhibitory concentrations of the samples having obvious inhibition effect at a concentration of 50 μg/disc were further tested in the same way. The tested samples and positive controls were dissolved in DMSO with a concentration of 1 or 10 mg/mL and then 2-fold diluted into a series of concentrations from 1 to 0.0078 mg/mL or from 10 to 0.078 mg/mL, respectively. Each paper disc loaded with 2.5 μL of diluted solution was placed on the inoculated plates. The plates were cultured at 30 °C for 2−5 days, and then the inhibited diameters of indicator fungi were measured. The lowest quality of each sample loaded on the disc that showed an obvious inhibition zone presented the antifungal potency. Antiviral Assay. HSV-1 strain and Vero cells were obtained from American Type Culture Collection. Cytotoxic activity was evaluated using Vero cell lines by an MTT method. Anti-HSV-1 activity was determined by a plaque assay using monolayer cultures of Vero cells in 24-well culture plates.18 Details are described in the Supporting Information.

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RESULTS AND DISCUSSION The molecular formula of simplicilliumtide I (1) was determined to be C28H38N4O6S by high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z 581.2399 [M + Na]+) and NMR data (Table 1). Its 1H and 13C NMR data showed similarity to the linear peptide simplicilliumtide B.10 The main differences between them were the absence of two 5117

DOI: 10.1021/acs.jafc.7b01238 J. Agric. Food Chem. 2017, 65, 5114−5121

Article

Journal of Agricultural and Food Chemistry

Figure 3. Key 1H−1H COSY and HMBC correlations of compounds 2−5.

had even been reported that the sulfoxide moiety would be partially reduced to sulfide moiety in the environment with high temperature and concentrated hydrochloric.23,24 It was reasonable to observe the existence of L-Met in the acidic hydrolysates of 1. The molecular formula of simplicilliumtide J (2) was determined as C46H73N7O11 by HRESIMS (m/z 900.5431 [M + H]+) and NMR data (Table 2). The 13C NMR displayed eight amide or ester carbonyl carbon signals at δC 173.6, 172.9, 172.1, 171.2, 170.8, 170.8, 170.8, 170.3. The 1H NMR spectrum revealed seven amide protons at δH 6.74 (1H, s), 7.22 (1H, s), 7.48 (1H, d, J = 9.0 Hz), 7.53 (1H, d, J = 8.0 Hz), 7.77 (1H, m), 7.79 (1H, m), 8.42 (1H, d, J = 8.0 Hz). These data displayed that 2 was a peptide. The overlapped signals at upfield δH 1.17−1.24 in the 1H NMR spectrum and a cluster of methylene carbon signals close to δC 28 in the DEPT 135 spectrum indicated a long-chain alkyl group existing in 2. Analysis of the HSQC, HMBC, and 1H−1H COSY spectra suggested that 2 contained six amino acid residues including an isoleucine (Ile), a tyrosine (Tyr), a glutamine (Gln), a proline (Pro), an alanine (Ala), and a threonine (Thr). The residual fragment was a long-chain fatty acid with 14 carbons. The downfield chemical shift of δC 73.5 (C-5, CH) indicated that C5 was an oxygenated methine carbon. The key 1H−1H COSY correlations from H-2 [δH 1.98 (1H, m), 2.14 (1H, m)] to H-3 [δH 1.31 (1H, m), 1.36 (1H, m)], and from H-4 [δH 1.33 (1H, m), 1.48 (1H, m)] to H-5 (δH 4.82, m), suggested that the long-chain fatty acid was a 5-hydroxytetradecanoic acid (HTA) residue. The HMBC and 1H−1H COSY correlations (Figure 3) suggested the sequence of the amino acid residues was Ile-TyrGln-Pro-Ala-Thr-HTA. Because the molecular formula required 14 degrees of unsaturation, it was deduced that the hydroxyl group on C-5 of HTA residue and the carboxyl group of Ile residue combined to form a lactone linkage, even though the HMBC correlation from H-5 [δH 4.82 (m)] of HTA to C-1 (δC 171.2) of Ile was not observed. The amino acid residues were identified as L-allo-Ile, D-Tyr, LGln, L-Pro, D-Ala, and D-allo-Thr by Marfey’s method17 and HPLC analysis (see Materials and Methods and Supporting Information). To determine the absolute configuration of C-5 in the 5-hydroxytetradecanoic acid fragment, the chemical conversion combined with Mosher’s method was used12 (see Materials and Methods). First, the phenolic hydroxyl group on Tyr was protected with methyl group to give 2a. Then the lactone bond in 2a was cleaved through a methanolysis reaction to give 2b that gave a HRESIMS ion peak at m/z 968.5686 [M + Na]+. Compound 2b was treated with (R)- and (S)-αmethoxy-α-(trifluoromethy)phenylacetyl chloride (MTPA-Cl)

upfield methyl signals and the appearance of a downfield methyl signal [δH 2.56 (3H, s), δC 37.7] in 1. Detailed analysis of the 2D NMR data (heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and correlation spectroscopy (COSY)) (Figure 2) revealed that 1 also contained the residues of an isoleucine (Ile), a N-methylphenylalanine (N-Me-Phe), and an 2-aminobenzoic acid (ABA), but did not contain a valine (Val) residue as simplicilliumtide B. For the residual unconfirmed signals, two methylenes of them appeared as two sets of signals in the 1H and 13C NMR spectra. One set was δH 2.09 (1H, m, H-3a), 2.31 (1H, m, H3b), 2.74 (1H, m, H-4a), 2.85 (1H, m, H-4b), δC 23.7 (CH2, C3), 49.2 (CH2, C-4). Another set was δH 2.09 (1H, m, H-3′a), 2.31 (1H, m, H-3′b), 2.74 (1H, m, H-4′a), 2.85 (1H, m, H4′b), δC 23.9 (CH2, C-3′), 49.3 (CH2, C-4′). These signals suggested that 1 might be a mixture of two isomers, which was supported by the HPLC analysis with a chiral column (CHIRALCEL OD-H 4.6 mm × 250 mmL, 5 μm; see Supporting Information) eluting with isopropanol/n-hexane (v:v 55:45, 1 mL/min), showing the presence of two peaks. In the HMBC spectrum, the correlations from the additional downfield methyl singlet [δH 2.56 (3H, s), δC 37.7] to the methylene carbons (δC 49.2/49.3, C-4/C-4′) suggested that the two groups might connect with a heteroatom having high electronegativity. According to the molecular formula of 1, the atom was speculated to be a sulfur atom. The 1H−1H COSY correlations from H-2 to NH/H-3, and from H-3 to H-4 (the above-mentioned protons), as well as comparisons of their chemical shifts with references19−22 and analysis of the HMBC data (Figure 2), suggested that 1 contained a methionine sulfoxide [Met (O)] residue. The isomerization of 1 was caused by the chirality of sulfoxide moiety. The methionine sulfoxide residue was probably formed by the oxidation of methionine residue during isolation.19 Furthermore, the HMBC correlations from NCH3 of N-MePhe to C-1 of Ile and C-2 of N-Me-Phe, and from NH of Met (O) to C-1 of N-Me-Phe and C-2 of Met (O), suggested that the sequence of the amino acid residues was Ile-N-Me-Phe-Met (O). The LR-ESIMS/MS (Figure 2) showed two major fragment ions at m/z 275.5 [Ile+N-Me-Phe]+ and 446.5 [NMe-Phe+Met (O)+ABA+2H]+, indicating the connection of ABA with Met (O) instead of Ile. The amino acid residues were identified as L-allo-Ile, D-N-Me-Phe, and L-Met (O), respectively, by Marfey’s method and HPLC analysis (see Materials and Methods). A FDAA derivate of L-Met was also observed in the HPLC chromatogram at 24.1 min, which was confirmed by the LR-LC-ESI-MS analysis (see Supporting Information). It 5118

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Figure 4. Chemical transformation flowchart of compounds 2 and 3.

Figure 5. Partial Δδ (δS − δR) values in ppm for MTPA esters of 2b and 3b.

3) suggested that 3 also contained six amino acid residues including a Val instead of an Ile, a Tyr, a Gln, a Pro, an Ala, and a Thr, and the sequence of the amino acid residues was ValTyr-Gln-Pro-Ala-Thr-HKTA. These amino acids were identified as L-Val, D-Tyr, L-Gln, L-Pro, D-Ala, and D-allo-Thr by Marfey’s method, respectively. The absolute configuration of C5 of HKTA was determined as S using the same methods as that of 2 (Figures 4 and 5). Simplicilliumtide L (4) exhibited the same molecular formula as 3 (C45H69N7O12) through analyzing HRESIMS (m/z 922.4893 [M + Na]+) and NMR data (Table 2). The 1H and 13 C NMR spectral data of 4 were greatly similar to that of 3. The HMBC spectrum of 4 showed correlations of H-11 (δH 2.37, m, 2H), H-13 (δH 2.39, m, 2H), and H-14 (δH 0.90, t, J = 7.0 Hz, 3H) with a keto carbonyl carbon (δC 210.9), along with the 1H−1H COSY spectrum showing correlations of H-13 with H-14 and H-10 with H-11 (Figure 3), which suggested the presence of a 5-hydroxy-12-ketotetradecanoic acid (HKTA) fragment. Further detailed analysis of the HSQC, HMBC, and 1 H−1H COSY spectra of 4 (Figure 3) suggested that 4 also contained the six amino acid residues of a Val, a Tyr, a Gln, a Pro, an Ala, and a Thr, and the sequence of the amino acid residues was Val-Tyr-Gln-Pro-Ala-Thr-HKTA. The amino acids were also identified as L-Val, D-Tyr, L-Gln, L-Pro, D-Ala, and Dallo-Thr by Marfey’s method, respectively. The absolute

in pyridine-d5, respectively, to yield the (S)- and (R)-MTPA ester derivatives 2c and 2d that showed quasi-molecular ion peaks at m/z 1394.6178 [M + Cl − H2O]− and 1394.6195 [M + Cl − H2O]− in their HRESIMS, respectively (Figure 4). Analysis of the 1H NMR data of 2c and 2d (see Supporting Information) and comparison of their Δδ (δS − δR) values (Figure 5) suggested the S configuration of C-5 in the 5hydroxytetradecanoic acid residue and the R configuration of C-3 in the Thr residue, which was consistent with the result of Marfey’s method. The molecular formula of simplicilliumtide K (3) was determined as C45H69N7O12 based on HRESIMS (m/z 922.4892 [M + Na]+) and NMR data (Table 2). The 1H, 13 C, and DEPT 135 NMR spectra of 3 were similar to those of 2, and the main differences between them were the additional presence of a keto carbonyl group at δC 208.5, the downfield shifts of a methylene [δH 2.38 (2H, t, J = 7.5 Hz), δC 42.6] and a methyl [δH 2.05 (3H, s), δC 29.6], and the absence of two methylene signals and a methyl group in 3. In the HMBC spectrum, the key correlations of H-12 (δH 2.38, t, J = 7.5 Hz) and H-14 (δH 2.05, s) with C-13 (δC 208.5, C), and H-14 with C-12 (δC 29.6, CH2), combined with other HMBC correlations and 1H−1H COSY correlations (Figure 3), suggested the presence of a 5-hydroxy-13-ketotetradecanoic acid (HKTA) fragment instead of a HTA fragment. Further detailed analysis of the HSQC, HMBC, and 1H−1H COSY spectra of 3 (Figure 5119

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bulged into a bubble-like structure and the number of spores was reduced significantly. For the inhibition zones with C. australiensis, the mycelial deformation and cell’s swelling were observed, and the mycelium grew slowly and produced pigment ahead of time. We speculated that the bioactive compounds 2, 6, and 7 did not influent the spores’ germination but changed the hyphal morphology, leading to influence on the growth and sporulation of the two strains. Compound 2a was more active against C. australiensis than 2, 6, and 7 because its inhibition zone was transparent. The MIC values (μg/disc) of 2, 6, and 7 toward A. versicolor and C. australiensis were close to that of the positive controls ketoconazole and amphotericin B; however, their inhibition zones were not as clear as the positive controls. Compounds 2−7 and their derivatives (2a−2d and 3a−3d) did not show growth inhibition toward A. sydowii and C. albicans at a concentration of 50 μg/disc. The antiviral activities of 1−7, 2a, and 3a toward HSV-1 were also evaluated using a plaque reduction assay. The results (Table 3) showed that, under their noncytotoxic concentrations (TC0) against a Vero cell line, 2, 6, and 7 had evident antiviral activity toward HSV-1 with IC50 values of 14.0, 16.7, and 15.6 μM, respectively. The TC0 and TC50 values against Vero cell line were 25.1, 204 μM for 2, 57.2, 137.0 μM for 6, and 49.4, 101.1 μM for 7, respectively, while other compounds showed no antiviral activity. The corresponding IC50 and TC0 values of positive control acyclovir were 3.0 and >1000 μM, respectively. Comparing the structures and bioactivities of 2−7 (Table 3), it seemed that the lactonized 5-hydroxytetradecanoic acid residue played an important role for the antifungal and antiviral activities of 2−4 and 6 and 7; if the lactone linkage was opened, their bioactivities would be lost. Furthermore, 2, 6, and 7 showed better antifungal and antiviral activities than 3 and 4, which suggested that the carbonyl substituent at C-13/C-14 of the 5-hydroxytetradecanoic acid residue could significantly reduce their bioactivities. The previous study had even discussed the correlation of the structure of amino acid and the antifungal activity among these kinds of cyclic peptides;8 however, this is the first time to discuss the effects of the lactone linkage and the substituent group of the fatty acid chain fragment on the bioactivity of this type of cyclic peptide. Also, this is the first time to report the antiviral activity of these cyclic peptides. Summarily, a new linear peptide 1 and four new cyclic peptides 2−5 together with known analogues 6 and 7 were isolated from the deep-sea-derived fungal strain S. obclavatum EIODSF 020. Also, a series of derivatives of 2 and 3 were obtained by methylation, methanolysis reaction, and Mosher reaction. The lactonization, ring opening, and diverse substitution of 5-hydroxytetradecanoic acid residue diversified the structures of these kinds of cyclic peptides, allowing for discussion of their structure−bioactivity relationships. Compounds 2, 6, and 7 showed significant antifungal activity toward A. versicolor and C. australiensis and also had evident antiviral activity toward HSV-1 with IC50 values of 14.0, 16.7, and 15.6 μM, respectively. Their structure−bioactivity relationships suggested that the exitence of the lactonized 5-hydroxytetradecanoic acid residue played an important role for the antifungal and antiviral activities of 2−4 and 6 and 7, and the substituent groups on the lactonized 5-hydroxytetradecanoic acid chain also could significantly affect the activities of 2−4 and 6 and 7. This is the first time to discuss the effects of the lactone linkage and the substituent group of the fatty acid chain fragment on the bioactivity of these types of cyclic peptides.

configuration of C-5 of HKTA in 4 was not further determined by Mosher method as 3 because of the lack of sufficient sample. However, according to the consistent optical rotation values ([α]25D − 12 for 3 and [α]25D − 10 for 4) and biosynthesis pathway of 3 and 4, we speculated the configuration of C-5 of HKTA in 4 was also S. The molecular formula of simplicilliumtide M (5) was assigned as C31H45N7O9 by HRESIMS (m/z 682.3172 [M + Na]+) and NMR data (Table 2). The 1H and 13C NMR spectral data of 5 were similar to that of 4, and the main difference was the disappearance of overlapped hydrogen signals at about δH 1.3 and a cluster of methylene carbon signals at about δC 28 in 5, which suggested that 5 did not contain an alkyl chain segment. Further comparison of the molecular formulas of 5 and 4 suggested that 5 lost a 5-hydroxy-12-ketotetradecanoic acid fragment with the formula of C14H24O3. Detailed analysis of the HSQC, HMBC, and 1H−1H COSY spectra of 5 (Figure 3) suggested that 5 also contained the six amino acid residues of a Val, a Tyr, a Gln, a Pro, an Ala, and a Thr, and the sequence of the amino acid residues was Val-Tyr-Gln-Pro-AlaThr. The amino acids were also identified as L-Val, D-Tyr, LGln, L-Pro, D-Ala, and D-allo-Thr by Marfey’s method, respectively. Previous studies reported that these kinds of cyclic peptides had antifungal activity.12−14 In here, the antifungal activities of 2−7 and their derivatives (2a−2d and 3a−3d) were also evaluated by disc diffusion method as reference.8 The results (Table 3) showed that 2, 6, and 7 showed significant inhibitory Table 3. Anti-HSV-1 and Antifungal Activities of Compounds 1−7, 2a−2d, and 3a−3d antifungal potency

(MIC, μg/disc)

samplea

anti-HSV-1 (IC50, μM)

A. versicolor

A. sydowii

C. australiensis

C. albicans

Kaz ApB 1 2 2a 2b 2c 2d 3 3a 3b 3c 3d 4 5 6 7

NT NT − 14.0 − NT NT NT − − NT NT NT − − 16.7 15.6

0.625b 0.156b NT 0.625c 25c − − − − − − − − − − 0.625c 1.562c

0.312b 0.020b NT − − − − − − − − − − − − − −

1.562b 0.156b NT 0.156c 0.312b 25c − − 50c − − − − 50c − 0.156c 0.156c

12.5b 0.312b NT − − − − − − − − − − − − − −

Kaz, ketoconazole; ApB, amphotericin B; “−”, no activity; NT, not tested. bThe zone of inhibition is transparent. cThe zone of inhibition is not transparent.

a

activity toward A. versicolor and C. australiensis, but their inhibition zones were not transparent, which indicated the mycelium could grow in the zones. The phenomena were further analyzed under an optical microscope to observe the hyphae growing in the inhibition zones (see Supporting Information, Tables S3 and S4). For the inhibition zones with A. versicolor, it was observed that some hyphae were 5120

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(11) Onishi, J. C.; Rowin, G. L.; Miller, J. E. Antibiotic A43F. U.S. Patent 4201771, May 6, 1980. (12) Ishidoh, K.; Kinoshita, H.; Igarashi, Y.; Ihara, F.; Nihira, T. Cyclic lipodepsipeptides verlamelin A and B, isolated from entomopathogenic fungus Lecanicillium sp. J. Antibiot. 2014, 67, 459−63. (13) Lee, D. W.; Kim, B. S. Antimicrobial cyclic peptides for plant disease control. Plant Pathol. J. 2015, 31, 1−11. (14) Rowin, G. L.; Miller, J. E.; Albersschonberg, G.; Onishi, J. C.; Davis, D.; Dulaney, E. L. Verlamelin, a new antifungal agent. J. Antibiot. 1986, 39, 1772−1775. (15) Ishidoh, K.-i.; Kinoshita, H.; Nihira, T. Identification of a gene cluster responsible for the biosynthesis of cyclic lipopeptide verlamelin. Appl. Microbiol. Biotechnol. 2014, 98, 7501−7510. (16) Zhang, X. Y.; Tang, G. L.; Xu, X. Y.; Nong, X. H.; Qi, S. H. Insights into deep-sea sediment fungal communities from the East Indian Ocean using targeted environmental sequencing combined with traditional cultivation. PLoS One 2014, 9, e109118. (17) Fujii, K.; Ikai, Y.; Mayumi, T.; Oka, H.; Suzuki, M.; Harada, K. A nonempirical method using LC/MS for determination of the absolute configuration of constituent amino acids in a peptide: Elucidation of limitations of Marfey’s method and of its separation mechanism. Anal. Chem. 1997, 69, 3346−3352. (18) Ma, S. C.; Du, J.; But, P. P. H.; Deng, X. L.; Zhang, Y. W.; Ooi, V. E. C.; Xu, H. X.; Lee, S. H. S.; Lee, S. F. Antiviral Chinese medicinal herbs against respiratory syncytial virus. J. Ethnopharmacol. 2002, 79, 205−211. (19) Chen, M.; Shao, C.-L.; Fu, X.-M.; Kong, C.-J.; She, Z.-G.; Wang, C.-Y. Lumazine peptides penilumamides B-D and the cyclic pentapeptide asperpeptide A from a gorgonian-derived Aspergillus sp fungus. J. Nat. Prod. 2014, 77, 1601−1606. (20) Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Nagle, D. G.; Paul, V. J. Symplostatin 2: a dolastatin 13 analogue from the marine cyanobacterium Symploca hydnoides. J. Nat. Prod. 1999, 62, 655−658. (21) Nogle, L. M.; Williamson, R. T.; Gerwick, W. H. Somamides A and B, two new depsipeptide analogues of dolastatin 13 from a Fijian cyanobacterial assemblage of Lyngbya majuscula and Schizothrix species. J. Nat. Prod. 2001, 64, 716−719. (22) Matthew, S.; Ross, C.; Paul, V. J.; Luesch, H. Pompanopeptins A and B, new cyclic peptides from the marine cyanobacterium Lyngbya confervoides. Tetrahedron 2008, 64, 4081−4089. (23) Scorrano, G. Equilibriums and reactions of organic sulfoxides in moderately concentrated acids. Acc. Chem. Res. 1973, 6, 132−138. (24) Shechter, Y. Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen-exchange between sulfoxide and sulfide. J. Biol. Chem. 1986, 261, 66−70.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b01238. Details of Materials and Methods, 1D/2D NMR and HRESIMS spectra of 1−7 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (86) 020-89022112. E-mail: [email protected]. ORCID

Shu-Hua Qi: 0000-0003-3802-3139 Funding

We are grateful for the financial support provided by the Strategic Leading Special Science and Technology Program of Chinese Academy of Sciences (XDA100304002), Natural Science Foundation of China (81673326 and 41376160), Regional Innovation Demonstration Project of Guangdong Province Marine Economic Development (GD2012-D01-002), and National Marine Public Welfare Research Project of China (201305017). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Zhi-Hui Xiao, Ai-Jun Sun, and Yun Zhang for their testing the NMR and MS data of the compounds. REFERENCES

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DOI: 10.1021/acs.jafc.7b01238 J. Agric. Food Chem. 2017, 65, 5114−5121