Cyclodepsipeptides and Other O-Containing Heterocyclic Metabolites from Beauveria felina EN-135, a Marine-Derived Entomopathogenic Fungus

Bioassay-guided fractionation of a culture extract of Beauveria felina EN-135, an entomopathogenic fungus isolated from a marine bryozoan, led to the isolation of a new cyclodepsipeptide, iso-isariin D (1); two new O-containing heterocyclic compounds that we have named felinones A and B (2 and 3); and four known cyclodepsipeptides (4–7). The structures were elucidated via spectroscopic analysis, and the absolute configurations of 1 and 2 were determined using single-crystal X-ray diffraction and CD, respectively. All isolated compounds were evaluated for antimicrobial activity and brine-shrimp (Artemia salina) lethality.


Introduction
Entomopathogenic fungi such as species from the genera Beauveria and Metarhizium have been frequently used as an alternative to chemical insecticides for agricultural pest control, and they are attracting increasing attention because of their ability to produce structurally unique and biologically active secondary metabolites [1]. The marine-derived fungal species Beauveria felina, which is poorly OPEN ACCESS described, has proven to be a rich source of various cyclodepsipeptides, such as the destruxin, isaridin, and isariin classes, as well as polyketides and terpenoids [2][3][4][5]. As part of our efforts toward the investigation of bioactive secondary metabolites of marine-derived fungi [6][7][8][9][10], Beauveria felina EN-135, an entomopathogenic fungus isolated from a marine bryozoan, attracted our attention because of the strong brine-shrimp lethality of the culture extract. Bioassay-guided fractionation of the EtOAc extract led to the isolation and identification of a new cyclodepsipeptide, iso-isariin D (1); two new O-containing heterocyclic compounds that we have termed felinones A and B (2 and 3); and four known cyclodepsipeptides: destruxin A (4), roseotoxin B (5), destruxin E chlorohydrin (6), and [β-Me-Pro] destruxin E chlorohydrin (7) (Figure 1). The brine-shrimp lethality and antimicrobial activity of these compounds were evaluated. Details of the isolation, structure elucidation, and biological activity of compounds 1-7 are reported herein.
Compound 1 is considered to be a cyclohexadepsipeptide of the isariin class [11][12][13], and a literature search revealed that the structure of this family of compounds has been primarily determined via spectroscopic analysis and the absolute configuration has been determined by analyzing the amino-acid derivatives, but none of these cyclohexadepsipeptides has been unambiguously determined via X-ray crystallography. We performed crystallization of 1 alongside the spectroscopic studies. Although compound 1 was initially obtained as a colorless amorphous powder, single crystals suitable for X-ray analysis were obtained after many attempts. The structure and absolute configuration of 1 could thus be further determined based on single-crystal X-ray diffraction using Cu Kα radiation ( Figure 3). The result not only confirmed the peptide sequence but also allowed the identification of the amino-acid residues as L-Val 3 , D-Leu 4 , L-Ala 5 , and L-Ala 6 , along with 2S-hydroxy-3S-methylpentanoic acid (HMA 1 ). Most reported cyclodepsipeptides in the isariin class have a 2-hydroxy aliphatic acid moiety characterized with a straight chain [11][12][13]; only one derivative, iso-isariin B, has a branch chain [4]. Compound 1 was the second cyclodepsipeptide of the isariin class to be identified that has this type of unusual HMA moiety, which differentiated it from isariin D (8). Thus, the structure of 1 was elucidated and named iso-isariin D. The HMA residue of iso-isariin D (1) in B. felina is most likely related to the polyketide biosynthesis pathway, and the amino-acid sequence might be obtained from the nonribosomal peptide biosynthesis pathway [14,15]. Compound 2 was obtained as a yellowish solid. The molecular formula C 11 H 16 O 4 was determined via HRESIMS, implying four degrees of unsaturation (one less than the reference compound 9) [16]. Detailed analyses of the 1D NMR data (Table 2) indicated the presence of one carbonyl carbon, three additional quaternary carbons (two sp 2 and one oxygenated sp 3 ), two oxygenated sp 3 methines, three methylenes (one oxygenated sp 3 ), and two methyl groups. Comparison between the NMR data of 2 and 9 indicated that their planar structures were very similar except that the signals of the double bond of C-8/C-9 at δ C 102.1 (CH) and 165.7 (C) in 9 were replaced by signals of a single bond at δ C 36.9 (CH 2 ) and 68.6 (CH) in the 13 C-NMR spectrum of 2. Accordingly, the proton signal at δ H 5.18 (s, H-8) of 9 disappeared in the 1 H NMR spectrum of 2. Instead, additional CH 2 and OCH signals at δ H 2.07 (m, H-8α), 2.20 (m, H-8β), and 3.54 (m, H-9) were observed in the 1 H-NMR spectrum of 2. The correlations from H-8 to H-9 and from H-9 to H-10 in the COSY spectrum of 2 supported the above deduction ( Figure 2).  The relative configuration of compound 2 was determined by analyzing the NOESY data and 1 H-NMR J-values [17]. The observed NOE correlations from OH-4 to H-5 and from 11-Me to H-6β indicated that they were on the same face of the molecule, respectively (Figure 2). However, the NOEs and coupling constants for H-6 and H-8 were difficult to measure due to overlap with other protons, but a spectrum in CD 3 OD afforded better resolution of these signals. (Table 2). The large coupling constants observed between H-5 and H-6β (9.5 Hz) and between H-9 and H-8α (10.1 Hz) indicated trans relationships. The key correlation between H-6α and H-8α in the NOESY spectrum measured in CD 3 OD finally established the relative configuration of 2 as shown in Figure 2.
The absolute configuration of 2 was determined by the application of CD. The CD spectrum displayed a negative Cotton Effect (CE) at 219 nm and a positive CE at 248 nm, which was nearly identical to that of (S)-2-acetyl-3,6-dihydroxycyclohex-2-enone, suggesting the presence of an S-configuration at C-4, which was opposite to that of pestafolide A [17,18]. The cyclohexenone ring of pestafolide A showed the same relative configuration as that of 2, but a reverse absolute stereochemistry, which was probably due to a different stereochemistry-selective biosynthetic pathway in Pestalotiopsis foedan [17]. The Mosher's method was also tried to further determine the absolute configuration of 2, but unsatisfied, probably due to the unstable MTPA esters. Therefore, the absolute configuration of 2 was tentatively assigned to be 4S, 5R, and 9R, and this compound was named felinone A.
Felinone B (3) was determined to have the molecular formula C 12 H 14 O 3 (six degrees of unsaturation) via HRESIMS. The 1D NMR spectra of 3 indicated the presence of four aromatic and one oxygenated sp 3 quaternary carbons, four sp 2 methines, one oxygenated sp 3 methylene, and two methyl groups ( Table 2). The structure of 3 was very similar to that of 10, a compound previously isolated from Smallanthus fruticosus [19]. The primary difference between compounds 3 and 10 was that the 13-Me and 12-OCH proton signals (δ H 1.54 and 4.99) of 10 [19] were absent in the 1 H NMR spectrum of 3 and, instead, an oxygenated methylene signal at δ H 4.65 (s, H-12) was observed. Detailed analyses of the COSY and HMBC spectra further confirmed the structure of compound 3 as shown in Figure 2.

Biological Activities of the Isolated Compounds
The isolated compounds 1-7 were evaluated for brine-shrimp lethality and antimicrobial activity. Among them, the hexadepsipeptides 1 and 4-7 exhibited potent lethality against brine shrimp (Artemia salina), with LD 50 values of 26.58, 5.34, 0.73, 2.16, and 1.03 μΜ, respectively, which were notably stronger than that of the positive control colchicine (with an LD 50 value of 88.4 μΜ). Compounds 2 and 3 exhibited weak activity with lethal rates of 61.4% and 59.6%, respectively, at a concentration of 100 μg/mL. The antimicrobial activities against six bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Vibrio alginolyticus, Vibrio anguillarum, and Edwardsiella tarda) and four plant pathogenic fungi (Physalospora piricola, Alternaria brassicae, Colletotrichum gloeosporioides, and Cucumber fusarium) were also evaluated. Only compound 3 showed inhibitory activity higher than that of the chloramphenicol control (MIC value of 4 μg/mL) against P. aeruginosa; this compound was found to have an MIC value of 32 μg/mL.

General
The optical rotations were determined using an Optical Activity AA-55 polarimeter. UV spectra were measured using a Lengguang Gold S54 spectrophotometer (Shanghai Lengguang Technology Co. Ltd., Shanghai, China). The 1 H, 13 C, and 2D NMR spectra were acquired using a Bruker Advance 500 spectrometer (Bruker BioSpin Group, Karlsruhe, Germany). Mass spectra were obtained using a VG Autospec 3000 mass spectrometer (VG instruments, London, UK). Semi-preparative HPLC was performed using a Dionex UltiMate

Fungal Material
The fungus Beauveria felina EN-135 was isolated from an unidentified marine bryozoan and identified via sequence analysis of the ITS region of its rDNA as previously described [22]. The sequence data derived from the fungus, which was similar (99%) to the sequence of Beauveria felina CBS 250.34 (compared to accession No. AY261369), was deposited in GenBank with the accession No. HQ891664. The strain was preserved at the Key Laboratory of Experimental Marine Biology at the Institute of Oceanology of the Chinese Academy of Sciences.

X-ray Crystallographic Analysis of Compound 1
The crystallographic data were collected using a Bruker D8-advance X-ray diffractometer (Bruker AXS Corporation, Karlsruhe, Germany) equipped with graphite monochromatic Cu-Kα radiation (λ = 1.54178 Å) at 293(2) K [23]. The absorption data were obtained using the program SADABS [24]. The structure was analyzed via direct methods using the SHELXTL software package [25]. All non-hydrogen atoms were refined anisotropically. The H atoms were located via geometrical calculations, and their positions and thermal parameters were fixed during structure refinement. The structure was refined using full-matrix least-squares techniques [26].

Brine-Shrimp Lethality
The brine-shrimp (Artemia salina) lethality of the isolated compounds was determined as previously described [27]. Colchicine was used as a positive control.

Antimicrobial Assay
The antibacterial activities against E. coli, S. aureus, P. aeruginosa, V. alginolyticus, V. anguillarum, and E. tarda along with the antifungal activities against P. piricola, A. brassicae, C. gloeosporioides, and C. fusarium were investigated using the disk diffusion and double dilution methods as previously described [28,29]. Chloramphenicol and amphotericin B were used as positive controls for the antibacterial and antifungal bioassays, respectively.

Conclusions
One novel cyclodepsipeptide, iso-isariin D (1), and two novel O-containing heterocyclic compounds, felinones A and B (2 and 3), were isolated from a culture of Beauveria felina EN-135. In addition, four known destruxin cyclodepsipeptides (4-7) were also identified. The structures were elucidated via spectroscopic analysis, and the absolute configurations of 1 and 2 were determined using single-crystal X-ray diffraction and CD, respectively. Compounds 1 and 4-7 exhibited potent brine-shrimp lethality with LD 50 values of 26.58, 5.34, 0.73, 2.16, and 1.03 μΜ, respectively, whereas compound 3 showed inhibitory activity against P. aeruginosa with an MIC value of 32 μg/mL.