Endophytic fungi are a fascinating source of new natural products which are of great potential for medicinal and agricultural applications [1,2,3,4]. Mangrove plants live in tropical and subtropical forests and share the ability to grow in estuarine and coastal environments. They are open systems with respect to both energy and biomatter and thus couple upland terrestrial and coastal estuarine ecosystems [5]. Mangrove-derived endophytes are attracting significant attention due to their potential of producing novel metabolites. Examples of those bioactive metabolites are the depsidone-type compound paeciloxocin A isolated from Paecilomyces sp., isolated from the bark of an unidentified mangrove from the Taiwan Strait, which was found to have potent cytotoxic activity [6]. Penicinoline was separated from a Penicillium sp., isolated from the bark of the mangrove Acanthus ilicifolius (Acanthaceae) collected from the South China Sea; it showed in vitro cytotoxic activity against 95-D and HepG2 cell lines [7]. Chloropupukeanolide A from Pestalotiopsis fici, obtained from branches of an unidentified mangrove in the suburb of Hangzhou, China, exhibited anti-HIV1 activity when tested in vitro [8].

During our ongoing search for new bioactive metabolites from plant-derived endophytes [9,10,11], we isolated an endophytic Bionectria ochroleuca strain from leaf tissues of the mangrove plant Sonneratia caseolaris (Sonneratiaceae), collected at Hainan Island in China.

Natural products of fungi belonging to the genus Bionectria were only rarely studied so far. A literature survey showed that Bionectria sp. yielded bionectriol A and TMC-151 which are both metabolites of polyketide origin [12,13]. Furthermore, piperazine derivatives bionectines A–C, glioperazine B and C and verticillin G were also reported from Bionectra byssicola [14,15,16]. In the present study we provide a comprehensive analysis of natural products produced by Bionectria ochroleuca and report on two new peptides designated pullularins E (1) and F (2), and two known congeners pullularins A (3) and C (4) [17], in addition to the fungal epipolythiodioxopiperazine metabolite verticillin D (5) [18].

The crude ethyl acetate extract of Bionectria ochroleuca cultured on solid rice medium, was taken to dryness and partitioned between n-hexane and 90% methanol. The 90% methanol fraction was chromatographed over different stationary phases (silica gel and Sephadex LH-20). Final purification by preparative reversed-phase HPLC afforded five compounds whose structures were elucidated by high resolution ESI mass spectrometry (HRESIMS) and NMR spectroscopy.

Pullularin E (1) was obtained as a white powder. Its UV maxima at 227 and 277 nm concurred with its colorless appearance. The molecular formula C₄₂H₅₇N₅O₈ was derived from the HRESIMS exhibiting a peak at m/z 760.4264 (calcd. for C₄₂H₅₈N₅O₈ 760.4285). After NMR data collection using CDCl₃, the HRESIMS of this compound showed different pseudomolecular peaks at m/z 794.3881 (100%, [M + H]⁺), and 796.3866 (32%, [M + 2 + H]⁺) indicating the molecular formula C₄₂H₅₆ClN₅O₈, containing one chlorine atom. Therefore, compound 1 was totally transformed to a chloro-derivative (1a). Compound 1a was re-measured in DMSO-d₆ to get better resolution of the NMR spectra. Extensive analysis of the NMR data of 1a and comparison with those reported for pullularins A (3) and C (4) [17] indicated a close structural relationship of 1a with the latter depsipeptides. The number of hydrogen and carbon atoms observed in the ¹H and ¹³C NMR spectra of 1a was in agreement with the molecular formula, indicating that 1a is a hexadepsipeptide composed of one 2-hydroxycarboxylic acid moiety and five amino acid residues. Correspondingly, one ester carbonyl carbon (δ_C 169.5) and five amide carbonyl carbons (δ_C 168.1, 165.4, 171.8, 173.8 and 168.0) were discernible. Since only two -NH proton signals (δ_H 9.53 and 8.89) and two N-methyl groups (δ_H 2.93 and 2.40) were observed in the ¹H NMR spectrum of 1a, the fifth amino acid was assumed to represent proline. This assumption was corroborated by analysis of the TOCSY spectrum, which in addition allowed for assigning the spin systems and furthermore hinted at the presence of an O-isoprenyl residue. The positions of the N-methyl groups, the prenyl residue, the sequence of the amino acid residues and the 2-hydroxycarboxylic acid were established by extensive analysis of the HMBC and ROESY data (Table 1).

The 2-hydroxycarboxylic acid was assigned to be 3-phenyllactic acid (3-Ph-Lac), on the basis of an oxymethine (δ_C 71.5) attached to a methylene at δ_C 35.8, which in turn was adjacent to a monosubstituted phenyl group. Extensive analysis of the NMR data showed that the serine residue in the known compound 3 was replaced by an alanine residue in 1a, as evidenced by the upfield shifted signals at δ_H 4.65 of the α-proton and at δ_H 1.21 of the aliphatic methyl group in the alanine residue. Its -NH group at δ_H 8.89 showed correlations to the methyl group and the carbonyl groups of proline, indicating that both amino acids were adjacent.

The NMR spectra of 1a differed from those of 3 and 4 furthermore by the nature of the isoprene unit. Instead of two olefinic methyl groups as present in compounds 3 and 4 only one signal was found for 1a at δ_H 1.80, sharing a COSY cross peak with the methylene function CH₂-4′ at (δ_H 5.20 and 5.04). The olefinic methyl proton signal also correlated with a quaternary carbon C-3′ at δ_C 141.3 and a methine carbon C-2′ at δ_C 63.0 in the HMBC spectrum. Moreover, a chlorine was assumed to be attached at C-2′, based on the chemical shifts of CH-2′ (δ_C 63.0, δ_H 4.86). Thus, 1a featured an O-isopentenyl moiety as isoprene substituent instead of an O-dimethylallyl residue as present in 3 and 4.

For the determination of the stereochemistry of the amino acid residues, the so-called advanced Marfey’s method was employed [19]. After hydrolysis of 1a in 6 M HCl for 24 h, the hydrolyzate was transformed to diastereomeric reaction products by adding the reagent FDAA. The reaction products thus obtained were submitted to analytical HPLC and LCMS. Moreover, the commercially available amino acids tyrosine, N-methyl-isoleucine, alanine, proline and N-methyl-alanine were used as authentic standards, both as the respective L-enantiomers and as racemates, and subjected to similar conditions for the derivatization and analysis. By comparison of the retention times of the reaction products of the hydrolyzate and the amino acid standards, the configuration of the respective amino acid was determined. As tyrosine lost its isoprenyl residue during hydrolysis, it was not necessary to provide O-isoprenyl tyrosine derivatives. On this basis, the configurations of the amino acids in 1a were determined as L-Tyr, N-Me-L-Ile, L-Ala, L-Pro and N-Me-L-Ala.

1a is obviously an artifact. Unfortunately, it was not possible to collect the NMR data of 1 in other deuterated solvents, since it was totally transformed to 1a. It was interesting that the transformation to a chloro-derivative was only observed in 1 despite that compounds 3 and 4 also have double bond in the isoprenyl residue and were also measured in CDCl₃. The structure of the new compound 1 was tentatively deduced as shown in Figure 1.

Pullularin F (2) exhibited a UV/VIS spectrum with λ_max at 226 and 275 nm, resembling those of the previously isolated pullularins. In the HRESIMS of 2 the pseudomolecular peak at m/z 709.3802 [M + H]⁺ indicated the molecular formula C₃₈H₅₂N₄O₉ (calcd. for C₃₈H₅₂N₄O₉ 709.3813), hinting to the presence of only four amino acids in the molecule. The compound could not be dissolved in CDCl₃ as for the other pullularins, but only in DMSO. Signals of the amino acid residues for proline, serine, N-methylated isoleucine and O-prenyl-tyrosine were readily observed, in the NMR spectra of 2 as well as signals for 3-phenyllactic acid, proving the absence of N-methylated alanine in 2. A conspicuous feature of the NMR spectra was the upfield shift of H-2 of 3-Ph-Lac to δ_H 4.10 as compared to other pullularins, indicating the presence of an alcohol function instead of an ester moiety. This finding and the lack of correlations between the α-hydroxycarboxylic acid and the O-prenyl-tyrosine indicated that pullularin F (2) was a linear and not a cyclic peptide. This finding was also consistent with the molecular formula obtained from HRESIMS. Moreover, it also explained the increase in polarity of 2 in comparison to pullularins A (3), C (4) and chloro-derivative of pullularin E (1a).

The peptide was hydrolyzed and the stereochemistry of the amino acids analyzed also via Marfey’s method [19]. On this basis, the configurations of the amino acids in 2 were determined as L-Tyr, N-Me-L-Ile, L-Ser and L-Pro.

All isolated compounds were subjected to a cytotoxicity assay employing the murine lymphoma L5178Y cell line, which is summarized in Table 2. Verticillin D (5) showed pronounced cytotoxic activities against the tested cell line. Antiproliferative properties were also prevalent among the cyclic depsipeptides pullularin A (3), C (4) and chloro-derivative of pullularin E (1a) with EC₅₀ values ranging between 0.1 and 6.7 µg/mL, whereas the linear pullularin F (2) did not exhibit any cytotoxic activity at the tested dose.

Pullularins E and F (1 and 2) together with three known compounds (3–5) were isolated from the EtOAc extract of the endophytic fungus Bionectria ochroleuca. Compounds 1a, 3, 4, and 5 exhibited pronounced to moderate cytotoxic activity against the mouse lymphoma cells (L5178Y) with EC₅₀ values ranging between 0.1 and 6.7 µg/mL.