New Ansamycins from the Deep-Sea-Derived Bacterium Ochrobactrum sp. OUCMDZ-2164

Two new ansamycins, trienomycins H (1) and I (2), together with the known trienomycinol (3), were isolated from the fermentation broth of the deep-sea-derived bacterium Ochrobactrum sp. OUCMDZ-2164. Their structures, including their absolute configurations, were elucidated based on spectroscopic analyses, ECD spectra, and Marfey’s method. Compound 1 exhibited cytotoxic effects on A549 and K562 cell lines with IC50 values of 15 and 23 μM, respectively.


Introduction
During the last two decades, increasingly more natural product (NP) chemists have set their sights on the sea because of the abundance of biological diversity of marine life. Numerous NPs with novel chemical skeletons and intriguing biological activities have been isolated from the marine organisms, including plants, animals, microorganisms, and so on [1][2][3]. Marine-derived microorganisms, as a huge resource for the discovery of active NPs, have attracted widespread attention [4][5][6][7][8][9][10][11]. Researchers tried to obtain new species from extreme environments, and the deep sea has become an important target [12][13][14][15], which was found to be a habitat with a diversity of species, especially microorganisms. The deep-sea-derived microorganisms have a great potential to produce new active compounds because of the special survival conditions [16][17][18][19]. For example, ammosamides A and B, produced by a deep-sea sediment-derived Streptomyces strain (1618 m), showed potent cytotoxicity against the HCT-116 cell line [20].
Ansamycins exhibit a broad range of bioactivities, such as antimicrobial [26], antitumor [27], and antiviral [28] activities. The macrocyclic system composed of an aromatic moiety embedded in an alicycle was a distinguishing feature of this class of compounds, attracting much attention from biosynthetic and chemical synthesis researchers [22,29,30]. Usually, ansamycins are classified as two types based on the aromatic moiety, which are, naphthalenic ones and benzenic ones. Naphthalenic ansamycins represented by rifamycin displayed strong antimicrobial activities, while some benzenic ansamycins showed strong anti-tumor activities [31].

Results and Discussion
Compound 1 was obtained as a yellow oil. Its molecular formula was determined as C 31 Figure S1), indicating 12 degrees of unsaturation. The UV spectrum of 1 showed similar absorptions with trienomycins at λ max 214, 254, 258, 272, and 284 nm [36]. The 1 H NMR spectrum (Table 1, Figure S2) showed three signals at δ H 6.30 (s), 6.43 (s), and 6.85 (s), as a result of a 1,3,5-trisubstituted benzene ring system. The conjugated triene signals at δ H 5.54−5.58 (2H, m, H-4 and H-9) and δ H 6.05−6.15 (4H, m, H-5, H-6, H-7, and H-8) were also observed in the 1 H NMR spectrum ( Table 1). The 13 C NMR revealed 31 carbon signals, which were classified by DEPT (distortionless enhancement by polarization transfer) and HSQC (heteronuclear single-quantum correlation) spectra as five methyl carbons (including a methoxy group), four methylene carbons, fifteen methine carbons (including 10 olefinic methine carbons), and seven quaternary carbons (including three carbonyls) ( Table 1, Figures S3-S5). Analysis of 1 H and 13 C NMR data revealed that compound 1 shared the same ansamycin-like ring system as that of trienomycinol (3) [36], which was supported by the COSY (correlation spectroscopy) correlations of Figure S6), together with the key HMBC (heteronuclear multiple bond correlation) correlations of 3-OCH 3 to C-3, H-2 to C-1, H-24 to C-13, H-15 to C-13/C-14, H-17 to C-18/C-19/C-23, H-19 to C-21, H-23 to C-19, H-21 to C-19/C-23, and 20-NH to C-1/C-19/C-21 ( Figure 2 and Figure S7). Careful comparison of the NMR data between 1 and 3 revealed observation of the signals of an N-acetylalanine in compound 1, which was confirmed by the COSY correlation of H-2 /H-3 , and the HMBC correlations of H-2 to C-1 /C-4 , 2 -NH to C-2 /C-4 , and H-5 to C-4 ( Figure 2). The connection of ansamycin-like ring moiety and N-acetylalanine unit was determined based on the HMBC correlation of H-11 to C-1 ( Figure 2). To determine the configuration of compound 1, the hydrolysis was carried out under acidic condition. The water-insoluble hydrolysate was identified as trienomycinol (3)   The similar ECD (electronic circular dichroism) Cotton effects of 1 to those of 3 further supported the absolute configuration of the ansa ring system (Figure 3). The absolute configuration of alanine unit was determined as D-by Marfey's method [40]. The 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) derivative of the acidic hydrolysate of 1 gave the same HPLC retention time as that of authentic D-Ala FDAA derivative ( Figure S14). Thus, compound 1 that we named trienomycin H was identified as 11-O-trienomycinol N-acetyl-D-alaninoate.   Only compound 1 showed selectively cytotoxic activity against A549 and K562 cell lines with the IC 50 values of 15 and 23 µM, respectively, while compounds 2 and 3 did not show cytotoxic activities against three tumor cell lines. The results indicated that the N-acylalanine ester moiety is required for the cytotoxicity, in accordance with the literature data [36].

Collection and Phylogenetic Analysis
The bacterial strain OUCMDZ-2164 was isolated from the deep-sea water sample collected from the South China Sea (2000 m depth) in April 2012. The sea water sample was collected by a conductance temperature depth (CTD) device (Qingdao, China). The sea water (3.0 mL) was deposited on agar plate (3 g/L beef extract, 20 g/L glucose, 10 g/L yeast extract, 10 g/L soluble starch, 10 g/L peptone, 2 g/L CaCO 3 , 0.5 g/L KH 2 PO 4 , and 0.5 g/L MgSO 4 , in seawater) containing nystatin (100 µg/mL) as a fungi inhibitor and incubated at 28 • C for 8 days, until a single colony appeared. The single colony was transferred into another agar plate. It was characterized as Ochrobactrum sp. according to its 16S rRNA gene sequences (GenBank accession No. KX394628).

Cytotoxic Assays
Cytotoxicity was assayed by the MTT [41] and CCK-8 [42,43] methods. In the MTT assay, A549 or MCF-7 cell line was grown in RPMI-1640 supplemented with 10% FBS under a humidified atmosphere of 5% CO 2 and 95% air at 37 • C, respectively. Cell suspension, 100 µL, at a density of 3 × 10 4 cell/mL was plated in 96-well microtiter plates, allowed to attach overnight, and then exposed to varying concentrations (10 −5 −10 −12 M) of compounds for 72 h. The MTT solution (20 µL, 5 mg/mL in IPMI-1640 medium) was then added to each well and incubated for 4 h. Old medium containing MTT was then gently replaced by DMSO and pipetted to dissolve any formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at 570 nm. In the CCK-8 assay, K562 cell line was grown in RPMI-1640 supplemented with 10% FBS under a humidified atmosphere of 5% CO 2 and 95% air at 37 • C. Cell suspension, 100 µL, at a density of 5 × 10 4 cell/mL was plated in 96-well microtiter plates and then exposed to varying concentrations (10 −5 −10 −12 M) of compounds after cultivation for 24 h. Three days later, 10 µL of CCK-8 solution was added 4 h before detection. Then, the absorbance (450 nm) was measured, and the growth rates of cells were computed. Adriamycin was used as the positive control with the IC 50 values of 1.00, 0.63, and 0.73 for the cell lines MCF-7, A549, and K562, respectively.

Conclusions
The present study revealed two new ansamycins, trienomycins H and I (1 and 2), from a deep-sea-derived bacterial strain, Ochrobactrum sp. OUCMDZ-2164. As the first example of ansamycin reported from a French soil-derived bacterium in 1950s [23], this is the first report of new ansamycins from the deep sea-derived bacteria. Trienomycin H (1) exhibited selectively cytotoxic effects on A549 and K562 cell lines with the IC 50 values of 15 and 23 µM, respectively. Combined with the data of trienomycins A-E against HeLa and P388 cell lines [36], our results supported that N-acylalanine ester moiety at C-11 is the key group responsible for the cytotoxic effects of trienomycins against tumor cell lines.
Author Contributions: Y.F. analyzed the data and prepared the draft of the manuscript; C.W. performed the most experiments; L.W. did the acidic hydrolysis; A.C. and P.P. tested the cytotoxic activity. P.F. checked the data and revised the manuscript; W.Z. designed and supervised the research and revised the final version.
Funding: This research received no external funding.