Three nitrogen-containing metabolites from an algicolous isolate of Trichoderma asperellum

Marine fungi have proven to be a large reservoir of structurally unique and biologically active compounds (Cao and Wang 2020; Liu et al. 2019). So far, over 2000 new natural products have been discovered from marine fungi, such as nitrogen-containing metabolites, sulfur-bearing compounds, and halogenated structures, even some of them with clinically relevant pharmacological activities (Jia et al. 2015; Meng et al. 2013; Newman and Cragg 2016; Rateb and Ebel 2011; Wang et al. 2018). The fungi inhabiting or associated with marine algae defined marine algicolous fungi have attracted a great attention for natural product researchers during the past two decades. At least 400 new secondary metabolites have been isolated from them up to now, including Aspergillus, Penicillium, Fusarium and Trichoderma (Nenkep et al. 2010; Sun et al. 2012; Tsuda et al. 2004). It has been discovered that Trichoderma fungi are almost ubiquitous in terrestrial and aquatic environments. To date, more than one hundred new terpenes, steroids, polyketides, alkaloids, and peptides with antimicroalgal, antibacterial, antifungal, cytotoxic, and enzyme-inhibitory properties have been obtained from the marine-derived Trichoderma species (Fang et al. 2019; Liang et al. 2016, 2019; Miao et al. 2012; Pang et al. 2018; Shi et al. 2018, 2019; Song et al. 2018a, b, c, d ; Su et al. 2018). Among them, T. asperellum is one of the most productive species, and its isolates from marine algae and sediments have contributed a number of new terpenes and nitrogen-bearing compounds that mainly comprise menthane, bisabolane, cyclonerane, harziane, ergostane, oxazole, diketopiperazine, and peptaibol derivatives (Ren et al. 2009; Shi et al. 2019; Song et al. 2018b, c, d ). The high novelty and diversity of metabolites from marine algicolous strains of T. asperellum have encouraged our continuous efforts to further explore them. A chemical investigation of the alga-derived endophytic T. asperellum A-YMD-9-2, originally separated and purified from the inner tissue of the marine marcroalga Gracilaria verrucosa, resulted in one new diketopiperazine, cyclo(L-5-MeO-Pro-L-5-MeO-Pro) (1), and two new nitrogen-containing cyclonerane sesquiterpene derivatives, 5′-acetoxy-deoxycyclonerin B (2) and 5′-acetoxy-deoxycyclonerin D (3) (Fig. 1). In this paper, the isolation, identification, and biological evaluation of these new metabolites are presented.

Figure 1.  Structures and key HMBC (arrow) and COSY (bold line) correlations of 1-3

Cyclo(L-5-MeO-Pro-L-5-MeO-Pro) (1) was obtained as colorless crystals. The molecular formula C₁₂H₁₈N₂O₄ was deduced on the basis of HREIMS data (m/z 254.1271 [M]⁺, calcd for C₁₂H₁₈N₂O₄, 254.1267), corresponding to five degrees of unsaturation. However, the ¹³C NMR spectrum (Table 1) only demonstrated the presence of six signals, including those for two methylenes, one methyl, two methines, and one quaternary carbon. Aided by HSQC correlations, all the ¹H NMR resonances (Table 1) were successfully assigned to the above protonated carbons. Compared to methylcordysinin A (Song et al. 2018c), 1 possesses the NMR data corresponding to a 5-methoxyproline residue. COSY correlations from H-2(2′) to H-5(5′) via H₂-3(3′) and H₂-4(4′) and HMBC correlations from H-2(2′) to C-1(1′), from H-5(5′) to C-2(2′) and C-3(3′), and from H-6(6′) to C-5(5′) confirmed the linkage of this residue (Fig. 1), and its relative configuration was demonstrated by the cross peak between H-2(2′) and H-6(6′) in the NOESY spectrum. In combination with the molecular formula and unsaturation requirements, 1 was proposed to be a symmetric diketopiperazine formed by the dimerization of two 5-methoxyprolines. Finally, the absolute configuration (1S, 5R, 1′S, 5′R) of 1 was ascertained by X-ray diffraction data (Fig. 2).

Figure 2.  X-ray crystallographic structure of 1 (numbered differently from the structure in the text)

5′-Acetoxy-deoxycyclonerin B (2) was isolated as a colorless oil. HREIMS (m/z 409.2821 [M]⁺, calcd for C₂₃H₃₉NO₅, 409.2828) analysis gave the molecular formula C₂₃H₃₉NO₅, implying five degrees of unsaturation. The ¹H NMR spectrum (Table 1) exhibited obvious resonances for one doublet and five singlets imputable to six methyls, two triplets attributable to two methylenes, one multiplet ascribed to a methine that was attached to a nitrogen or oxygen atom, and three singlets assignable to double-bond protons. Twenty-three signals appeared in the ¹³C NMR spectrum (Table 1), ascribed to six methyls, seven methylenes, four methines, and six nonprotonated carbons by DEPT and HSQC data. The above NMR data were very similar to those of deoxycyclonerin B (Song et al. 2019), except for the existence of an extra acetyl group, confirmed by the HMBC correlations from H₃-8′ and H₂-5′ to C-7′ (Fig. 1). The cross peaks of H₃-1/H-2/H-6/H₂-5/H₂-4, H₂-8/H₂-9/H-10 and H₂-4′/H₂-5′ in the COSY spectrum, coupled with cross peaks from H₃-1 to C-2, C-3, and C-6, from H₃-13 to C-2, C-3, and C-4, from H₃-14 to C-6, C-7, and C-8, from H₂-12 and H₃-15 to C-10 and C-11, from H-10, H-2′, and NH to C-1′, and from H₃-6′ to C-2′, C-3′ and C-4′ in the HMBC spectrum, further defined the planar structure of 5′-acetoxy-deoxycyclonerin B (Fig. 1). On account of the NOE correlation between H-2′ and H₂-4′ and the similar NMR data with those of deoxycyclonerins (Song et al. 2019), the E-geometry was appointed to the vinyl group at C-2′. The relative configurations at C-2, C-3, C-6, and C-7 were certified by the identical ¹H and ¹³C NMR data with those of cyclonerane derivatives (Langhanki et al. 2014; Laurent et al. 1990; Song et al. 2018b). The absolute configurations at C-2, C-3, C-6, and C-7 were deduced to be uniform with its analogues according to biogenic consideration (Song et al. 2019). To corroborate the absolute configuration of C-10, the electronic circular dichroism (ECD) spectrum of 1 was measured in methanol, which showed a negative Cotton effect at 225 nm (Fig. 3). In light of the biosynthetic origin and agreements of experimental ECD spectra between 2 and deoxycyclonerin B (Song et al. 2019), the stereogenic centers of 5′-acetoxy-deoxycyclonerin B were determined to be 2S, 3R, 6R, 7R, and 10R.

Figure 3.  ECD spectrum of 2 in MeOH

An analysis of HREIMS (m/z 409.2830 [M]⁺, calcd for C₂₃H₃₉NO₅, 409.2828) data ascertained the molecular formula of 5′-acetoxy-deoxycyclonerin D (3) to be C₂₃H₃₉NO₅, the same as for 2. The ¹H and ¹³C NMR data (Table 1) of 3 resembled those of 2, apart from the resonances for the side chain unit, which were confirmed by the COSY correlations of H₂-8/H-9/H-10 and HMBC correlations from H₃-12 and H₃-15 to C-10 and C-11 as displayed in Fig. 1, including a 9-cycloneren-3, 7, 11-triol residue and an acetylated trans-anhydromevalonyl group (Anke et al. 1991; Song et al. 2018b). Furthermore, the HMBC interactions from NH to C-11, C-12, C-15, and C-1′ validated the connectivity of these two moieties. The large spin coupling constant (J=15.7 Hz) of H-9 and H-10 and the correlation between H-2′ and H₂-4′ in the NOE spectrum suggested the vinyl groups at C-9 and C-2′ to be E configuration. The absolute structure of 3, named 5′-acetoxy-deoxycyclonerin D, was regarded as that of 2 on the basis of biosynthetic origin.

Compounds 1-3 were evaluated for their inhibitory activities of four marine phytoplankton species (Chattonella marina, Heterosigma akashiwo, Karlodinium veneficum, and Prorocentrum donghaiense) (Shi et al. 2019). As shown in Table 2, these compounds have effects on the four phytoplankton species tested, with EC₅₀ values ranging from 12.7 to 351 μmol/L. In addition, an Artemia salina assay was carried out to detect the marine zooplankton toxicity of 1-3, but no lethality was measured at 100 μg/ml (Miao et al. 2012). The inhibition of marine-derived bacteria Vibrio parahaemolyticus, V. anguillarum, V. harveyi, V. splendidus, and Pseudoalteromonas citrea by 1-3 were also assayed, though no inhibition was observed at 40 μg/disk.

The spectra of IR were detected on a Nicolet iS10 FT-IR instrument (Thermo Fisher Scientific, Waltham, MA, USA). The spectra of UV and ECD as well as optical rotations were recorded on a Chirascan CD instrument (Applied Photophysics Ltd., Surrey, UK). NMR spectra (1D and 2D) were measured on a Bruker Avance III 500 NMR instrument (Bruker Corp., Billerica, MA, USA). Low and high resolution EI mass spectral data were obtained on an Autospec Premier P776 mass instrument (Waters Corp., Milford, MA, USA). Thin-layer chromatography was operated with precoated silica gel plates (GF-254, Qingdao Haiyang Chemical Co., Qingdao, China). Column chromatography was implemented with silica gel (200-300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China), RP-18 (AAG12S50, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden).

The fungal strain (GenBank no. MH819724), fermentation, extraction, and inceptive isolation were described in previous work (Song et al. 2019). The third fraction was chromatographed on a Si gel column with petroleum ether/ethyl acetate (2:1) and was further isolated by column chromatography on RP-18 (methanol/water, 3:7) and preparative thin-layer chromatography (dichloromethane/methanol, 50:1) to afford 1 (118 mg). The fourth fraction eluted with petroleum ether/ethyl acetate (1:1) on a Si gel column and was further purified by column chromatography on RP-18 (methanol/water, 1:1 to 3:2) and Sephadex LH-20 (methanol) and preparative thin-layer chromatography (dichloromethane/methanol, 15:1) to get 2 (52.0 mg) and 3 (2.0 mg).

Cyclo(L-5-MeO-Pro-L-5-MeO-Pro) (1): colorless crystals; mp 169-171 ℃; [α]_D²⁰ - 102 (c 0.94, MeOH); IR (KBr) v_max 2957, 2937, 2836, 1688, 1667, 1452, 1378, 1207, 1080, 864 cm^-1; ¹H and ¹³C NMR data, refer to Table 1; EIMS m/z (%) 254 [M]⁺ (19), 239 (11), 224 (100), 195 (39), 100 (45), 68 (44), 58 (33), 42 (31); HREIMS m/z 254.1271 [M]⁺, calcd for C₁₂H₁₈N₂O₄, 254.1267 (Supplementary Figs. S1-S7).

5′-Acetoxy-deoxycyclonerin B (2): colorless oil; [α]_D²⁰ - 13 (c 2.6, MeOH); IR (KBr) v_max 3426, 2963, 1728, 1667, 1634, 1532, 1452, 1373, 1247, 1172, 1043, 919, 886 cm^-1; ¹H and ¹³C NMR data, refer to Table 1; EIMS m/z (%) 409 [M]⁺ (1), 391 (6), 374 (5), 334 (4), 296 (53), 252 (16), 224 (48), 164 (22), 113 (100), 95 (41), 83 (90), 44 (42); HREIMS m/z 409.2821 [M]⁺, calcd for C₂₃H₃₉NO₅, 409.2828 (Supplementary Figs. S8-S14).

5′-Acetoxy-deoxycyclonerin D (3): colorless oil; [α]_D²⁰ - 21 (c 0.12, MeOH); IR (KBr) v_max 3436, 2965, 1726, 1664, 1636, 1529, 1450, 1383, 1243, 1169, 1044, 975, 920 cm^-1; ¹H and ¹³C NMR data, refer to Table 1; EIMS m/z (%) 409 [M]⁺ (0.5), 392 (3), 374 (8), 296 (12), 253 (37), 172 (68), 139 (27), 113 (64), 82 (47), 44 (100); HREIMS m/z 409.2830 [M]⁺, calcd for C₂₃H₃₉NO₅, 409.2828 (Supplementary Figs. S15-S21).

X-ray diffraction data were collected on a Bruker Smart-1000 CCD diffractometer with a graphite-monochromatic Cu-Kα radiation (λ=1.54178 Å) at 293(2) K. These data were corrected for absorption through the program SADABS (Sheldrick 1996). The structure was elucidated by direct methods with the SHELXTL software package (Sheldrick 1997a) and polished by full-matrix least-squares techniques (Sheldrick 1997b). All non-hydrogen atoms were refined anisotropically, while the hydrogen atoms were placed by using geometrical calculations and their positions and thermal parameters were settled in the process of the structure refinement.

Crystal data for 1: C₁₂H₁₈N₂O₄, M_r 254.28, monoclinic, space group: P2₁2₁2₁, unit cell dimensions a=6.8497 (4) Å, b=10.3453 (10) Å, c=17.6854 (13) Å, V=1253.23 (17) ų, α=β=γ=90°, Z=4, d_calcd=1.348 Mg/m³, crystal dimensions 0.23×0.18×0.05 mm, μ=0.847 mm^-1, F(000)=544.0, measured independent reflections=1835, number of reflections used for refinement=1571, parameters used for refinement=166, and Lorentz and polarization corrections were used. The final refinement showed R₁=0.0440 and wR₂=0.1275 [I > 2σ(I)]. The Flack parameter was 0.1(4) and these data have been submitted to the Cambridge Crystallographic Data Centre, with deposition No. CCDC 1865401.

The inhibition of marine phytoplankton Chattonella marina, Heterosigma akashiwo, Karlodinium veneficum, and Prorocentrum donghaiense and the toxicity to the marine zooplankton Artemia salina were tested as described previously (Miao et al. 2012; Shi et al. 2019), and K₂Cr₂O₇ was chosen as a positive control. The antibacterial activity against Vibrio parahaemolyticus, V. anguillarum, V. harveyi, V. splendidus, and Pseudoalteromonas citrea were assayed at 40 μg/disk as stated previously (Miao et al. 2012).

In summary, chemical investigation of the marine-alga-endophytic fungus T. asperellum A-YMD-9-2 has generated the isolation and identification of three new compounds (1‒3), which greatly enriches the diversity of nitrogen-containing metabolites. Among the new structures, 2 and 3 feature better inhibition of H. akashiwo and P. donghaiense, respectively, and display no toxicity to any of the zooplankton assayed.

This work is fnancially supported by the National Natural Science Foundation of China (No. 31670355), the Natural Science Foundation for Distinguished Young Scholars of Shandong Province (No. JQ201712), and the Youth Innovation Promotion Association of the CAS (No. 2013138). NJ gratefully acknowledges the support of Taishan Scholar Project Special Funding (tsqn201909164).

NJ conceived and designed the experiments; YS carried out the tests; FM and XY contributed reagents/materials/ analysis tools; YS and NJ wrote the paper. All authors have read and approve of the manuscript.

The authors declare that they have no confict of interest.

This article does not contain any studies with human participants or animals performed by any of the authors.