Levantilides A and B, 20-Membered Macrolides from a Micromonospora Strain Isolated from the Mediterranean Deep Sea Sediment

Two new 20-membered macrolides, levantilide A and B, were isolated from the Micromonospora strain M71-A77. Strain M71-A77 was recovered from an Eastern Mediterranean deep-sea sediment sample and revealed to produce the levantilides under in situ salinity of 38.6‰. The chemical structures of the levantilides were elucidated on the basis of different one- and two- dimensional NMR experiments. Levantilide A exhibits a moderate antiproliferative activity against several tumor cell lines.


Introduction
The deep sea is an extreme environment which is still marginally investigated and harbors a great variety of bacteria that have, so far, not been cultivated. Bacteria which live in the deep sea need to adapt to the specific environmental characteristics such as high hydrostatic pressure, low temperature and only occasional nutrient supply. These constraints quite likely determine the phylogenetic diversity of the deep-sea bacterial communities and also affect the secondary metabolite production of these bacteria. Therefore, deep-sea bacteria are considered as a promising source for the discovery of new natural products. Marine members of Actinobacteria are highly potent producers of interesting compounds [1][2][3][4][5] as was already shown for their terrestrial counterparts [6]. Only recently, two strains of Streptomyces sp. from the Atlantic ocean deep-sea sediment were shown to produce the two new natural products caboxamycin and albidopyrone [7,8].
With special focus on the discovery of new natural products, we selectively isolated Actinobacteria from the deep-sea sediment of the Eastern Mediterranean Sea (the so-called Levantine Sea). This environment is characterized by a relatively high bottom temperature of 13-14 °C, salinity values of approximately 38-39‰, high hydrostatic pressure (440 bar at the sampling site) and an extreme depletion of nutrients [9]. Among the isolated bacteria, strain M71-A77 produced two new macrolides named levantilides A (1) and B (2), which will be described in this paper.

Results and Discussion
Strain M71-A77 was isolated from the Eastern Mediterranean deep-sea sediment (4400 m) and revealed 99.3% 16S rRNA gene sequence similarity to Micromonospora auratinigra DSM 44815 T (AB159779). Analyses of the culture extract of this strain (incubated in liquid soja-peptone medium) by HPLC-DAD-MS led to the detection of two unknown 20-membered macrolides, levantilide A (1) and B (2), with detected masses of m/z 508 and m/z 506, respectively. Subsequent cultivation of the strain in larger scale (10 L) led to the isolation of the levantilides as colorless solids.  (2), showed a mass difference of 2 amu in the HPLC-DAD-MS measurement, which indicated one additional double bond, which for example can be observed, when a hydroxy-group is replaced by a carbonyl function, as it was the case here. Already in the 1 H NMR spectrum it was obvious that all signals belonging to protons of the macrolide ring were identical in both molecules (see Table 2). However, significant differences could be observed for the signals of the side chain. Analysis of the data showed that the methine group CH-23 was no longer present in levantilide B. Instead of it, an additional signal of a carbonyl carbon appeared, its shift of 210.5 ppm proving it to be a ketone. As a consequence of the presence of a carbonyl group instead of a methine in position 23, the signal of H 2 -24 was no longer a multiplet, but appeared as a quartet as it only coupled with the methyl group CH 3 -25. Thus, the planar structure of 2 was established as depicted.     The levantilides are macrolides with a 20-membered lactone ring. From a biosynthetic point of view macrolides are typical type I PKS products with very well studied biosynthetic pathways. From the structures of the levantilides, a very simple assembly of a propionate starter unit, five further propionate building blocks and altogether six acetate building blocks can be deduced.
According to Skropeta (2008), polyketide metabolites have been reported from all water depths, but interestingly only 8% of the marine natural products known so far are produced by organisms obtained at depth greater than 1000 m [23]. As a matter of course, this might be due to the fact that the deep sea is hardly accessible. In the present study, it was shown by cultivation of strain M71-A77 with habitat sea water (38.6‰) that levantilides are also produced under the high salinity conditions occurring in situ in the Mediterranean Sea. Though strains of Micromonospora spp. were frequently isolated from deep sea habitats [24][25][26], to the best of our knowledge the levantilides are the first natural products described from a Micromonospora sp. strain isolated from the deep sea.

Isolation and identification of strain M71-A77
Strain M71-A77 has been isolated from a sediment core (1.5-5 cm sediment horizon) from 4400 m depth during a research cruise with RV Meteor M71/2 in the Eastern Mediterranean Sea, the so-called Levantine Sea [34° 25.48 N, 26° 05.39 E]. One gram of the sediment sample was transferred to a sterile petri dish and dried for 2 months at 20 °C prior to incubation for 1 h at 120 °C dry heat.
Sediment was then re-suspended in demineralized water and inoculated on agar plates of XJ4-medium containing of 1 L of demineralized water 18 g agar, 0.1 g histidine, 1 g raffinose, 0.5 g sodium hydrogen phosphate, 1.7 g potassium chloride, 0.05 g magnesium sulfate, 0.01 g iron sulfate, 0.02 g calcium carbonate, 0.5 mg thiamine hydrogen chloride, 0.5 mg riboflavine, 0.5 mg niacine, 0.5 mg piridoxin, 0.5 mg calcium pantothenate, 0.5 mg inositol, 0.5 mg para aminobenzoic acid and 0.25 mg biotin. After 2 months of incubation at 28 °C , strain M71-A77 was isolated by transferring to fresh XJ4-medium. The strain was classified by 16S rRNA gene sequence analysis according to Gä rtner et al. [27]. The 16S rRNA gene sequence was deposited in the EMBL Nucleotide Sequence Database and was assigned the accession no. FR714833.

Chemical analysis
General experimental procedures. The optical rotation was measured on a Perkin Elmer model 241 polarimeter. UV-spectra were obtained on a NanoVue (GE Healthcare). NMR spectra were recorded on a Bruker DRX500 spectrometer (500 and 125 MHz for 1 H and 13 C NMR, respectively), using the signals of the residual solvent protons and the solvent carbons as internal references (δ H 2.04 and δ C 28.9 ppm for acetone-d 6 ; δ H 2.50 and δ C 39.51 ppm for DMSO-d 6 ). High-resolution mass spectra were acquired on a benchtop time-of-flight spectrometer (MicrOTOF, Bruker Daltonics) with positive electrospray ionization. Analytical reversed phase HPLC-UV/MS experiments were performed using a C 18 column (Phenomenex Onyx Monolithic C18, 100 × 3.00 mm) applying an H 2 O (A)/MeCN (B) gradient with 0.1% HCOOH added to both solvents (gradient: 0 min 5% B, 4 min 60% B, 6 min 100% B; flow 2 mL/min) on a VWR Hitachi Elite LaChrom system coupled to an ESI-ion trap detector (Esquire 4000, Bruker Daltonics). Preparative HPLC was carried out using a Phenomenex Gemini C18 110A AXIA, 100 × 50.00 mm column.

Isolation of levantilides A and B.
10 L of liquid starch-peptone medium (1L demineralized water 10 g starch, 5 g soja peptone, 15 g Tropic Marin® sea salt and 1 g calcium carbonate) were used for cultivation of strain M71-A77. After 8 days of incubation (28 °C, 125 rpm), the culture supernatant was separated from the cells by centrifugation at 10,000 rpm for 10 min (Beckman J2-MC). Cell pellets were suspended in methanol and homogenized three times with an Ultra Turax T25 basic (IKA-Werke GmbH & Co., Staufen, Germany) at 17.500 U/min for 1 min. After additional centrifugation, the methanol extract was decanted and dried. The culture broth supernatant was extracted with ethylacetate (1:1). The dried extracts were dissolved in methanol and analyzed by HPLC-UV/MS. Levantilides A and B were detected at 4.2 and 4.5 min with a maximum UV-absorption at 260 nm. For structure analysis, 1 and 2 were separated by reversed phase HPLC. For that purpose, HCOOH (0.1%) was added to the solvents H 2 O (A) and MeCN (B) and a gradient from 10% B over 60% B (reached after 17 min) to 100% B was applied (flow 15 mL/min). Levantilides A and B were detected at 16.6 and 17.8 min. Thus, 7 mg of 1 and 3 mg of 2 were obtained.  1H, m, H-13a Table 2; HRESIMS m/z 529.3509 [M + Na] + (C 30 H 50 NaO 6 , 529.3500).

Production of levantilide A (1) and B (2) at in situ salinity
Strain M71-A77 was tested for the production of secondary metabolites at habitat salinity (38.6 ‰). For that purpose, Tropical Marine® salt and aqua dest. were replaced by Mediterranean Sea water obtained from the sampling site. After 8 days of cultivation at 28 °C, the culture broth was extracted with ethylacetate and analyzed by analytical HPLC-UV/MS as described above.

Cytotoxic tests
The in vitro antiproliferative activities of compound 1 against the gastric cancer cell line GXF 251L, lung cancer cell line LXFL 529L, melanoma cancer cell line MEXF 462NL, mammary cancer cell line MAXF 401NL, renal cancer cell line RXF 486L and pancreatic cancer cell line PAXF 1657L were determined by Oncotest GmbH (Freiburg, Germany) using a modified propidium iodide monolayer assay [29]. Compound 2 was not tested by Oncotest GmbH, for there was not enough left of the compound.