Oleamycins A and B: new antibacterial cyclic hexadepsipeptides isolated from a terrestrial Streptomyces sp.

Description

Actinomycetes have been a source for anti-infectives for more than 50 years, and continue to have a very important role as the source organisms for the discovery of new antibiotics. In the course of our screening program for the discovery of new antibacterial compounds, one such strain (Lv20-58), recovered from the root zone of the plant Oleaceae europea came to our attention.

The strain (Lv20-58), later identified to belong to the genus Streptomyces was cultivated in M medium (6 l) for 8 days at 30 °C and then extracted with ethyl acetate (6 l) to give a crude extract of 138.9 mg. The crude extract was partitioned between 20 ml of hexane, CH₂Cl₂ and MeOH to afford 49.8, 80.9 and 10.6 mg fractions, respectively. The CH₂Cl₂ fraction was subsequently purified by semipreparative reverse-phase HPLC to yield compounds 1 (t_R=30.5 min; 2.5 mg) and 2 (t_R=28.6 min, 0.4 mg). A detailed account of the spectroscopic analysis leading to the assignment of structures to oleamycin A (1) and oleamycin B (2) are presented below.

HR-ESI(+)MS analysis of oleamycin A (1) (Table 1) revealed a pseudomolecular ion ([M+Na]⁺) indicative of a molecular formula (C₃₉H₆₆N₈O₁₁Na) requiring 11 double bond equivalents. The NMR (CDCl₃) (Table 2) data revealed resonances for seven ester/amide carbonyls (δ_C 169.2–177.1) requiring 1 to incorporate four rings. Early on in the structural elucidation phase we noticed the presence of two distinct set of resonances in the NMR (Table 2). As a result, we focused on the interpretation of the major set of resonances that led to the structural assignment of oleamycins. The 39 carbons and their assorted proton resonances were attributed to 2 primary methyls (δ_C 9.6 and 11.9), 3 secondary methyls (δ_C 18.3, 19.6 and 18.9), 3 tertiary methyls from which 2 were attributed to N-methyls (δ_C 33.4 and 34.3), 14 methylenes (δ_C 20.0–52.3), 7 methines (δ_C 29.9–82.1) and 9 quaternary carbons (δ_C 77.4–177.1)). Further analysis of the NMR data (Table 2, Figure 1) proposed that 1 was a new cyclic hexapeptide consisting of a 3-hydroxyleucine residue, three glycine residues (two of which were N-methylated) and two piperazic-acid residues. The sequence of the amino acids was determined by interpretation of key correlations observed in the HMBC spectra. Specifically, HMBC correlations from the methine H-3 (δ_H 4.62) and methylene H₂-8 (δ_H 4.28, 3.63) to the ester carbonyl C-7 (δ_C 170.6). Additional correlations from N-Me (δ_H 2.83) and the methylene H₂-10 (δ_H 5.32, 3.36) to the amide carbonyl C-9 (δ_C 169.3), extended by similar correlations from N-Me (δ_H 3.07) and the methylene H₂-12 (δ_H 4.54, 3.69) to the amide carbonyl C-11(δ_C 169.2) linked the N-Me-glycine residues 2 and 3. The amide NH (δ_H 7.53) of the third glycine residue and the methine H-14 (δ_H 5.15) of the piperazic-acid residue (Pip1) showed correlations to C-13 (δ_C 169.8 ), whereas H-14 and H-19 (δ_H 5.50) showed correlations to C-18 (175.0). Finally, correlations from H-3 and H-19 to C-1, led to the construction of the 19-membered cyclic hexapeptide fragment (Figure 1).

Table 1 Physicochemical properties of 1 and 2

Table 2 NMR (500 MHz, CDCl3) data for 1

Figure 1

Key 2D NMR (500 MHz, CDCl₃) correlations for 1 and Newman projection showing relative configuration of C2/C3.

The second structural feature of 1 was identified to be a polyketide side chain (Figure 1). The ¹H NMR (500 MHz, CDCl₃) (Table 2, Figure 1) and COSY data revealed a single isolated spin system, a primary methyl H₃-35 (δ_H 0.84) with correlations to a methylene H₂-34 (δ_H 1.09–1.24), methine H-33 (δ_H 1.30–1.44), secondary methyl H₃-36 (δ_H 0.98) and a methylene H₂-32 (δ_H 0.98). A second primary methyl H₃-31 (δ_H 0.79) extended to a methylene H₂-30 (δ_H 1.25–1.37), a deshielded methine H-29 (δ_H 3.53, δ_C 76.1) and finally to the methylenes H₂-27 (1.30–1.38) and H₂-26 (1.61–0.1.76). In the HMBC spectrum, a tertiary methyl H₃-37 (δ_H 1.27) showed correlations to an amide carbonyl C-23 (δ_C 177.1), oxycarbon C-24 (δ_C 77.4) and hemiketal carbon C-25 (δ_C 99.1). The absence of a hydroxyl signal associated with C-29 suggested that it has an ether linkage, which was attributed to the likely presence of a heterocyclic system. This was supported by HMBC correlations from the methylene H₂-26 (δ_H 1.61–1.76) to C-25, suggesting the presence of a tetrahydropyran ring. A large coupling (J_29,28=10.1 Hz) established a trans-diequatorial relationship orientation of the substituents at C-28 and C-29. The point of attachment of the polyketide unit to the peptide was established on the basis of HMBC correlations of the amide 2N-H (δ_H 8.00) and the tertiary methyl H₃-37 (δ_H 1.27) to the amide carbonyl C-23 (δ_C 177.1), leading to the overall planar structure of 1 (Figure 2). The relative configuration of the two stereogenic centers in β-OH-Leu was established as 2S* and 3S* by ROESY and J-based configuration analysis¹ (Figure 1). Despite the absence of a ROESY correlation from the methine H-4 to 2N-H, we have drawn the C2-C3 rotamer as follows, based on the absolute configuration identified as (2S, 3S) of the β-OH-Leu residue in the known 19-membered cyclic depsipeptides (A83586C, L-156,602, aurantimycins, polyoxypeptins, GE3 and dentigerumycin; Figure 2). HRESI(+)MS analysis of oleamycin B (2) (Supplementary Table S1) revealed a pseudomolecular ion ([M+Na]⁺) indicative of a molecular formula (C₃₈H₆₄N₈O₁₁Na) requiring 11 double bond equivalents (Table 1). The principle difference of 2 over 1 was the substituent on the tetrahydropyran ring from an isobutyl to an isopropyl residue (C32-C-35) (Figure 2). The closest known natural product analogs to 1 and 2 are the rare class of 19-memebered cyclic hexadepsipeptides azinothricin,² A83586C,³ L-156,602,⁴ citropeptin,⁵ variapeptin,⁶ verucopeptin,⁷ aurantimycins,⁸ polyoxypeptins,⁹ piplamycin,¹⁰ IC101,¹¹ GE3¹² (Figure 2) and the recently reported dentigerumycin.¹³ Oleamycin A (1) displayed significant biological activity against a panel of Gram-positive bacteria and a cancer cell line (HCT-116). It is noteworthy that MIC’s were against strains S. aureus (0.23 μg ml⁻¹) and Micrococcus luteus (0.03 μg ml⁻¹), whereas an IC₅₀ of 6.5 ng ml⁻¹ was recorded against HCT-116 cells (human colon carcinoma). Owing to the limited supply of oleamycin B (2) and reproducibility issues in the fermentation, we were unable to screen for its biological activity. In summary, we have isolated, characterized and evaluated the biological activity of two new members, oleamycin A (1) and B (2), of the well-described 19-membered cyclic depsipeptides.

Figure 2

Structures of 1,2 and related metabolites.

Experimental procedure

NMR spectra were obtained on a Bruker Ascend 500 MHz spectrometer equipped with a cryoprobe system (Bruker Biospin GmbH, Rheinstetten, Germany) in the solvents indicated and referenced to residual ¹H signals in deuterated solvents. ESI-MS were acquired using an Agilent 1100 Series separations module equipped with an Agilent 1100 Series LC/MSD mass detector (Agilent, Waldbronn, Germany) in both positive and negative ion modes under the following conditions: Zorbax C₈ column (Crawford Scientific, Lanarkshire, UK), 150 × 4.6 mm², eluting with 0.4 ml min⁻¹ 95% H₂O/MeCN to 5% H₂O/MeCN (with isocratic 0.01% trifluoroacetic acid) over 22 min, and then held for 5 min. HR-MS was carried out using an UltiMate 3000 rapid separation LC system (Dionex RSLC, Idstein, Germany) coupled to an ultra-high resolution time-of-flight MS (Bruker Daltonik MaXis, Bremen, Germany) operating in the positive ESI mode.

Sampling was performed in the Nikitsky Botanical Garden of Crimea (Ukraine). The soil (1 g) was collected from the root zone of O. europea and resuspended in sterile water, followed by serial dilutions leading to the inoculation onto the oatmeal agar (oatmeal 40 g l⁻¹, agar 15 g l⁻¹, pH 7.5). The plates were incubated for 20 days at 28 °C. Individual colonies were transferred onto new oatmeal agar plates for further analysis and cryopreservation. The 16S ribosomal DNA sequence analysis of strain Lv20-58 classified it to belong to the genus Streptomyces. The strain Streptomyces sp., Lv20-58, is deposited in the microorganism collection of Ivan Franko Lviv National University.

Strain Lv 20-58 was cultivated in a (250 ml) Schott flask containing M1 (1% starch, 0.4% yeast extract and 0.2% peptone) prepared in distilled water (80 ml). The strains were shaken at 150 r.p.m. for 8 days at 30 °C, extracted with EtOAc (50 ml) and the organic phase was concentrated in vacuo to yield a crude extract of 3.4 mg. The crude extracts were redissolved in MeOH generating a concentration of 1 mg ml⁻¹ and analyzed using HPLC-DAD-ESI(±)MS.

Five 5 l Erlenmeyer flasks containing M1 broth (1.2 l) were inoculated with starter culture (20 ml) of Streptomyces sp. The flasks were incubated at 30 °C on a rotary shaker at 150 r.p.m. for 8 d, extracted with EtOAc (2 × 500 ml² per flask) and the organic phases were concentrated in vacuo to yield a combined EtOAc extract (167.6 mg). The EtOAc extract was sequentially triturated with hexane, CH₂Cl₂ and MeOH (40 ml aliquots), which were concentrated in vacuo, to yield 37.8, 77.9 and 30.6 mg partitions, respectively. The CH₂Cl₂ soluble material was further fractionated by HPLC (Zorbax, C₈ column, 250 × 9.4 mm², 5 μm, 3 ml min⁻¹, gradient from 10 to 100% acetonitrile–H₂O over 30 min, with a 100% acetonitrile hold for 5 min) to afford oleamycin A (1) (t_R=30.5 min, 1.6 mg) and oleamycin B (2) (t_R=28.6 min, 0.4 mg).