Introduction

Fungi growing in special environments tends to produce bioactive secondary metabolites with plenty of structure characteristics because of their evolutional and differential metabolic systems that highly adapted during the process of natural selection.1, 2, 3, 4 In recent years, many bioactive compounds were discovered from Ophiocordyceps-colonizing fungi and Ophiocordyceps-related fungi isolated from the soil sample surrounding the fruiting body of O. sinensis, which inhabits low temperature and high altitude environments.5, 6, 7, 8 In our course of investigating new bioactive natural products from the above fungi, a strain S. trichophoricola (P068) was isolated from the soil sample in Maqin Country, Qinghai Province, People’s Republic of China, which surrounded the fruiting body of O. sinensis. Some noteworthy examples of bioactive compounds have been found from the Stagonospora genus: elsinochrome A and leptosphaerodione,9 two phytotoxin polyketides isolated from Stagonospora convolvuli (LA39); stagonolide H,10 a toxin from fungal pathogen Stagonospora cirsii; dihydromaldoxin,11 an endothelin receptor antagonist from Stagonospora sp.; and pramanicin,12 an antimicrobial agent from Stagonospora sp.

Through chemical investigation of the extract from the fermentation of S. trichophoricola, five new peptides, trichopeptide A (1), a linear tetrapeptide, trichopeptide B (2), a linear tripeptide and trichocyclodipeptides A–C (3–5), and three diketopiperazines were obtained. Details of the isolation, structure elucidation and antimicrobial activities of these compounds are reported herein.

Results and Discussion

Trichopeptide A (1) was obtained as a brown gum. It has a molecular formula of C27H42N4O7 (nine degrees of unsaturation), on the basis of its HR–ESI–MS pseudomolecular ion peak ([M+H]+ at m/z 535.3124, calcd for 535.3126). Analysis of its 1H and 13C NMR data (Table 1) revealed the presence of three amide N-H protons (δH 7.59, 7.73 and 8.45), six methyl groups including one N-methyl (δH 3.11 and δC 31.1), four methylenes, six methines including three N-methines, one monosubstituted benzene ring and five carboxylic carbons (δC 168.9, 170.4, 171.1, 173.0 and 175.0). These data accounted for all the nine degrees of unsaturation. Interpretation of COSY of 1 identified five proton spin systems, which were C-2-C-6, NH-C-γ (homoserine, Hse), NH-C-β (alanine, Ala), C-α-C-δ/C-δ′ (N-methyl leucine, N-MeLeu) and NH-C-δ/C-γ′ (isoleucine, Ile). Interpretation of its 1H, 13C NMR and HMBC data, the amino acid residues of Hse, Ala, N-MeLeu and Ile were established. The amino acid sequence of 1 was deduced to be Ile-N-MeLeu-Ala-Hse by analyses of the HMBC correlations of NH (δH-Ile 7.73) to C (δC-N-MeLeu 170.4), N-Me (δH-N-MeLeu 3.11) to C (δC-Ala 173.0) and NH (δH-Ala 8.45) to C (δC-Hse 171.1). The HMBC correlations from protons H-2/H-6 (δH 7.82) and the N-H of Hse (δH 7.59) to C-7 (δC 168.9) showed the connection of the linear peptide to the benzoyl group. Thus, the planar structure of trichopeptide A (1) was established (Figure 2).

Table 1 NMR spectral data of compounds 1 and 2
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Marfey’s method was applied to identify the absolute configurations of the amino acid residues derived from the acid hydrolysis of 1. The 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide derivatives of acid hydrolyzate of 1 and the corresponding D-configuration and L-configuration standards were subjected to HPLC–MS analysis. The absolute configurations of the amino acid residues of 1 were confirmed by comparison of the HPLC retention times and molecular mass with the corresponding standards. The amino acid residues Hse, N-MeLeu and Ile units were determined to have the L-configuration, whereas the Ala unit was determined to have the D-configuration (Supplementary Table S1). Thus, the structure of trichopeptide A (1) was established (Figure 1).

Figure 1
figure 1

Structures of compounds 1–5.

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Trichopeptide B (2) was obtained as a brown gum. It has a molecular formula of C21H31N3O6 (eight degrees of unsaturation) by HR–ESI–MS pseudomolecular ion peak ([M+H]+ at m/z 422.2295, calcd for 422.2286). Analysis of its 1H and 13C NMR data (Table 1) revealed a structure similar to 1, as one benzoyl connected to the Hse-Ala-N-MeLeu sequence. According to 113 of molecular mass less than 1, the Ile residue was missing. The Ile residue is replaced by a hydroxyl (chemical shift of carboxylic carbon in N-MeLeu: δC 170.4 in 1, δC 173.8 in 2). Thus, the planar structure of 2 was proposed (Figure 2). The absolute configurations of the Hse and N-MeLeu residues in 2 were assigned as L-configuration and the Ala unit was assigned as D-configuration through Marfey’s method (Supplementary Table S1). The structure of trichopeptide B (2) was established as shown (Figure 1).

Figure 2
figure 2

Key 1H-1H COSY, HMBC correlations of compounds 1–5.

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Trichocyclodipeptide A (3) was isolated as white amorphous powder. It has the molecular formula of C26H40N4O8 (nine degrees of unsaturation) determined by HR–ESI–MS pseudomolecular ion peak ([M+H]+ at m/z 537.2926, calcd for 537.2919). The 13C NMR data of 3 showed 13 carbon resonances. In accordance with the molecular formula, we speculated that 3 had a completely symmetrical structure. The 1H and 13C NMR data (Table 2) of 3 revealed four methyl groups, ten methylenes including two O-methylene, four methines, four olefinic carbons and six carboxylic carbons. These data accounted for eight degrees of unsaturation. The structure of 3 was similar to eleutherazine B,13 with a diketopiperazine skeleton. The difference between them was that the hydroxyl groups of eleutherazine B at C-10 and C-10′ are acetylated in 3. Interpretation of the COSY of half side of the structure of 3 revealed two proton spin-systems of C-2-C-5 and C-9-C-10. The substructure of C-1-C-5 was deduced as Orn residue by analysis of its NMR data. The HMBC correlations from olefinic proton H-7 (δH 5.68) to C-6 (δC 169.4) and C-8 (δC 150.7) indicated that C-6-C-8 was anα, β-unsaturated carbonyl. The correlations from H2-9 (δH 2.39) and H3-13 (δH 2.09) to C-7 (δC 121.2) indicated that the C-9 and C-13 (δc 18.4) were attached to C-8 and the moiety of C-6-C-10/C-13 was established. The correlations from H2-5 (δH 3.19) and H-7 (δH 5.68) to C-6 (δC 169.4) indicated that C-5 (δC 39.5) and C-7 are connected by amide linkage. The chemical shift value of C-10 (δC 63.2) and the HMBC correlations from H3-12 (δH 1.98) to C-11 (δC 172.7) and H2-10 (δH 4.17) to C-11 (δC 172.7) indicated that an acetyl group linked to C-10 through an oxygen atom. The chemical shift of C-2 (δC 55.7) and the HMBC correlations from H-2 (δH 3.96)/H-3 (δH 1.81) to C-1 (δC 170.4) indicated a lactone moiety. Comparing NMR data with leutherazine B indicated the existence of a cyclic dipeptide skeleton. Thus, the planar structure of 3 was established (Figure 2).

Table 2 NMR spectral data of compound 3 in CD3OD
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Trichocyclodipeptide B (4) was obtained as white amorphous powder. It has the molecular formula of C24H38N4O7 (eight degrees of unsaturation) determined by HR–ESI–MS pseudomolecular ion peak (M+H]+ at m/z 495.2816, calc dfor 495.2813). Most of the signals appeared in pairs, it was easy to deduce that the structure of 4 was similar to 3. Compound 4 is 42 of molecular mass less than 3 and the chemical shifts of C-10′ shifted upfield (δH 4.17, δC 63.2 in 3, δH 2.50, δC 59.1 in 4), an acetyl group was missing in 4, whereas rest of the substructure of 4 was corresponding to 3.

Trichocyclodipeptides C (5) was obtained as white amorphous powder. It has the molecular formula of C20H32N4O6 (seven degrees of unsaturation) determined by HR–ESI–MS pseudomolecular ion peak ([M+H]+ at m/z 425.2397, calcd for 425.2395). Analysis of its NMR data (Table 3) revealed the moiety of C-6′-C-10′/C-11′ in 4 was replaced by an acetyl group (δH/C 1.78/22.6; 169.0 in 5). These results were confirmed by the HMBC correlations of from H3-7′ (δH 1.78) and H2-5′ (δH 3.00) to C-6′. Rest of the substructure of 5 was corresponding to 4.

Table 3 NMR spectral data of compounds 4 and 5 in DMSO-d6
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The absolute configurations of the Orn residues in 3–5 were assigned as L-configuration through Marfey’s method (Supplementary Table S1). Thus, the structures of 3–5 were established as shown (Figure 1).

Compounds 1–5 were tested for antimicrobial activity against the fungus C. albicans (CGMCC 2.2086), the Gram-positive bacterium Staphylococcus aureus (ATCC 6538), the Gram-negative bacterium Escherichia coli (ATCC 1.0090) and the Bacillus subtilis (ATCC 6663), respectively (Table 4). Compound 3 showed antifungal activity against C. albicans with the IC50 and MIC values of 22 and 90 μg ml−1, respectively. The positive control amphotericin showed the IC50 and MIC values of 5.32 and 18.13 μg ml−1, respectively. Compounds 2 and 5 showed the antibacterial activity against S. aureus (vancomycin: IC50=0.08, MIC=0.54 μg ml−1) with the IC50 values of 45 and 44 μg ml−1, respectively. Compounds 3 and 5 showed the antibacterial activity against B. subtilis (streptomycin: IC50=0.13, MIC=0.32 μg ml−1) with the IC50 values of 46 and 45 μg ml−1, respectively.

Table 4 Antimicrobial activities of compounds 1–5
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Compounds 1–5 were also tested for cytotoxic activity for two cancer cell lines including lung cell (A549) and leukemia cell (K562), evaluated with the CCK8 cell viability assay. Compound 4 has weak cytotoxicity against K562 with IC50 value of 132 μg ml−1, whereas the positive control taxol showed IC50 value of 0.92 μg ml−1.

Although linear peptides have been isolated frequently,14, 15, 16 such as antibiotic-BK-230 from Burkholderia glumae, celenamides A and B from Cliona celata, compounds 1 and 2 distinctly differ from most of the known natural products. The presence of a D-Ala amino acid residue, only a few ones contained D-Ala amino acid residue, such as microcystin-LR from Microcystis spp., have been previously reported.17 The compounds containing D-Ala amino acid residue were reported to possess growth inhibition bioactivity against some bacteria.18 Diketopiperazines, such as eleutherazine B from the traditional Chinese medicine plant Acanthopanax senticosus, metachelins A and B from the insect-pathogenic fungus Metarhizium robertsii were discovered recently,19 and alternarizines A and B from an endophytic fungus Alternaria alternata and talarazines A–E, from an Australian mud dauber wasp-associated fungus, Talaromyces sp.20, 21. However, little antimicrobial activity was reported, the new structures 3–5 showed the moderate antimicrobial activities.

Materials and methods

General experimental procedures

Optical rotations were measured on an Anton Paar MCP200 polarimeter (Graz, Austria). 1H and 13C NMR data were acquired with Bruker Avance-500 spectrometers (Rheinstetten, Germany). The HSQC and HMBC experiments were optimized for 125.0 and 8.0 Hz, respectively. HR–ESI–MS and HPLC–ESI–MS data were recorded on an Accurate-Mass-Q-TOF LC/MS 6520 instrument (Santa Clara, CA, USA) in positive ion mode. HPLC data were obtained with a Waters 2695 instrument (Milford, MA, USA). Preparative HPLC was performed on an Agilent 1200 HPLC system using a C18 column (7.8 × 300 mm, Waters, 7 μm; detector: UV) with a flow rate of 2.2 ml min−1. The absorbance of contents in the 96-well clear plate was detected by a SpectraMax Paradigm microplate reader (Sunnyvale, CA, USA).

Fungal material

The strain of S. trichophoricola was isolated from a soil sample surrounding the fruiting body of O. sinensis collected in Maqin Country, Qinghai Province, People’s Republic of China, in 2014. The isolated strain was identified by morphology and sequence analysis (Genbank Accession Number KY750315) of the rDNA internal transcribed spacer (ITS) region. The strain was firstly cultivated on culture dish of potato-dextrose agar at 25 °C for 10 days. Then the agar was cut into grain size to inoculate in two conical flasks (500 ml) that each containing 300 ml autoclave sterilized potato-dextrose broth. The flasks containing inoculated potato-dextrose broth were cultivated at 25 °C on a rotary shaker at 170 rpm for 5 days. Thirty 500 ml Fernbach flasks, each containing 80 g of rice and 100 ml of distilled water, were autoclaved at 120 °C for 30 min, in which the fermentation proceeded. After cooling to room temperature, each of the flasks was inoculated with 10 ml of the spore inoculum and cultured at 25 °C for 30 days.

Extraction and isolation

The ferment culture was extracted with EtOAc (three times, each 6 l) and vacuum-dried to afford the crude extract (~10 g). The extract was fractionated by silica gel vacuum liquid chromatography (VLC) in petroleum ether-Acetone-MeOH gradient elution (Supplementary Figure S1). The fraction (2 g) eluted with 10% MeOH was then loaded on a silica gel column (2.5 × 45 cm) eluted with CH2Cl2-MeOH. The fraction (538 mg) eluted from 20:1 CH2Cl2:MeOH was isolated by Sephadex LH-20 column chromatography then eluted with MeOH, the subfractions were selected and purified by RP HPLC (Waters Symmetry Prep C18 column; 7 μm; 7.8 × 300 mm; 60–70% MeOH in H2O with 0.1% HCOOH for 45 min; 2.2 ml min−1) to afford 1 (10.0 mg, tR19 min) and 2 (6.0 mg, tR 17.00 min) (Supplementary Figure S2). The fraction (368 mg) eluted from 10:1 CH2Cl2:MeOH was isolated by Sephadex LH-20 column chromatography eluted in MeOH. The subfractions were purified by RP HPLC (Waters Symmetry Prep C18 column; 7 μm; 7.8 × 300 mm; 40–60% MeOH in H2O with 0.1% HCOOH for 50 min; 2.2 ml min−1) to afford 3 (10.0 mg, tR 27.20 min), 4 (8.0 mg, tR 20.50 min) and 5 (6.0 mg, tR 23.00 min) (Supplementary Figure S2).

Compounds characterization

Trichopeptide A ( 1): Brown gum; [α]D−9.0 (c 0.1, MeOH); UV (MeOH) λmax 224 nm (log ɛ) (3.98); NMR data (500 MHz, CDCl3) see Table 1; HR–ESI–MS m/z 535.3124 [M+H]+ (calcd for C27H43N4O7 535.3126).

Trichopeptide B ( 2): Brown gum; [α]D−48.0 (c 0.1, MeOH); UV (MeOH) λmax 210 nm (log ɛ) (3.86); NMR data (500 MHz, Acetone-d6) see Table 1; HR–ESI–MS m/z 422.2295 [M+H]+ (calcd for C21H32N3O6 422.2286).

Trichocyclodipeptide A ( 3): White powder; [α]D−22.0 (c 0.05, MeOH); UV (MeOH) λmax 222 nm (log ɛ) (3.76); NMR data (500 MHz, CD3OD) see Table 2; HR–ESI–MS m/z 537.2926 [M+H]+ (calcd for C26H41N4O8 537.2919).

Trichocyclodipeptide B ( 4): White powder; [α]D−14.0 (c 0.05, MeOH); UV (MeOH) λmax 215 nm (log ɛ) (3.83); NMR data (500 MHz, dimethyl sulfoxide-d6) see Table 3; HR–ESI–MS m/z 495.2816 [M+H]+ (calcd for C24H39N4O7 495.2813).

Trichocyclodipeptide C ( 5): White powder; [α]D−18.0 (c 0.1, MeOH); UV (MeOH) λmax 210 nm (log ɛ) (3.67); NMR data (500 MHz, dimethyl sulfoxide-d6) see Table 3; HR–ESI–MS m/z 425.2397 [M+H]+ (calcd for C20H33N4O6 425.2395).

Absolute configuration

Compounds 1–5 (1 mg each) with 6 N HCl (2.0 ml) were heated at 115 °C for 10 h.22 Then the hydrolyzates were placed in a 1 ml reaction vial added 60 μl 1.0 N NaHCO3 and 150 μl 1% solution of 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide in acetone. The vials were heated at 45 °C for 2 h, after cooling to room temperature, adding 30 μl 2.0 N HCl. The corresponding L- and D- standards were derivatized in the same was. The derivatives of the hydrolyzates and the standards were subjected to HPLC–MS analysis (Hypersil GOLD C18 column; 5 μm, 4.6 × 250 mm; 1.0 ml min−1) at 30 °C in gradient program: linear gradient 15–45% MeCN in H2O with 0.1% HCOOH for 50 min at 340 nm UV detection. The retention time data were recorded (Supplementary Table S1).

Antimicrobial assay

The antimicrobial assay was following the recommendations from the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards (NCCLS))23, 24 and conducted in triplicate. The fungal strain, C. albicans (CGMCC 2.2086), was grown in potato-dextrose broth, whereas the bacterial strain, S. aureus (ATCC 6538), B. subtilis (ATCC 6663) and E. coli (ATCC 1.0090) were grown in lysogeny broth. The targeted microbe was cultivated in broth at 25 °C for 48 h (fungi) and at 37 °C for 24 h (bacteria), till the final suspension reached 106 cells ml−1. Test samples (10 mg ml−1 as stock solution in dimethyl sulfoxide and serial dilutions) were transferred to a 96-well clear plate in triplicate, and the suspension of the test organism was added to each well, achieving a final volume of 100 μl (amphotericin, vancomycin, streptomycin and kanamycin were used as the positive control). After incubating at 25 °C for 48 h (fungi) and at 37 °C for 24 h (bacteria), the absorbance was detected with a microplate reader under 595 nm. The inhibition was calculated and plotted vs test concentrations to afford the IC50 and MIC.

Cytotoxicity bioassay

Cytotoxic activity for cancer cell lines including A549 and K562 cell lines was evaluated with the CCK8 cell viability assay.23 After treating cells with the compounds tested (in dimethyl sulfoxide) for 48 h in 96-well plates, adding 3 μl of CCK8 medium solution to each well, then culturing the tumor cells at 37 °C in a humidified atmosphere of 5% CO2 air for 4 h. The plate was read by microplate reader under 450 nm. The inhibition was calculated and plotted vs test concentrations to afford the IC50 in triplicate.