Halichoblelide D, a New Elaiophylin Derivative with Potent Cytotoxic Activity from Mangrove-Derived Streptomyces sp. 219807

During our search for interesting bioactive secondary metabolites from mangrove actinomycetes, the strain Streptomyces sp. 219807 which produced a high elaiophylin yield of 4486 mg/L was obtained. A new elaiophylin derivative, halichoblelide D (1), along with seven known analogues 2–8 was isolated and identified from the culture broth. Their chemical structures were determined by detailed analysis of 1D and 2D NMR and HRMS data. The absolute configuration of halichoblelide D (1) was confirmed by comparing the CD spectrum with those of the reported analogues. Compounds 1–7 exhibited potent cytotoxic activities against HeLa and MCF-7 cells with IC50 values ranging from 0.19 to 2.12 μM.


Introduction
There is a continuous and urgent demand for iterative cycles of drug discovery and development to combat new emerging human diseases and multi-drug resistant pathogens [1,2].Microbial natural products have delivered a plethora of structurally diverse specialized metabolites with potent pharmaceutical properties [3].Microorganisms derived from underexplored habitats, such as mangrove ecosystems, are especially noteworthy, in part owing to their unique biosynthetic potential and the corresponding attenuation of the likelihood of finding high numbers of known compounds [4][5][6].Mangrove ecosystems are extensively distributed in the interface between terrestrial and marine environments at tropical and subtropical latitudes, thereby suffering from extremely harsh stresses, including temperature, salinity, moisture and anoxia [7].Therefore, mangrove-derived actinomycetes are distinct from terrestrial isolates in physiology and genotype to cope with the rigorous environments.Several decades of studies have demonstrated that actinomycetes recovered from mangrove systems are increasingly recognized as a vast reservoir of new bioactive natural products, such as salinosporamide A [8], xiamycins [9], and streptocarbazoles A and B [10].Accordingly, our studies are focused on the mangrove ecosystem actinomycetes.

Yield of Elaiophylin in Shake-Flask Fermentation under Laboratory Conditions
Strain 219807 has been cultured on 18 different media available in our lab, and was found to produce the highest yield of elaiophylin on DO medium (50 mL in 250 mL Erlenmeyer flask), the yield of elaiophylin (4) measured by HPLC was calculated based on its standard curve [25].The regression equation of elaiophylin was obtained as y = 630x ´1.6007, and the calibration curve showed good linearity (R 2 = 0.9993) (Figure S11).According to the equation, the yield of elaiophylin reached 4486 mg/L in shake-flask fermentation under laboratory conditions, which was significantly more (>2 fold) than the highest yield reported before [26].This high yield was primarily dependent on the strain of the microorganism.The proper medium DO which contained a complex carbon sources as observed by other reports [27,28] also contributed to the high production.

Cytotoxic Activity of Compounds 1-7
Compounds 1-7 showed potent activities against human cervical carcinoma HeLa and breast cancer MCF-7 cell lines with IC 50 values ranging from 0.19 to 2.12 µM (Table 2).Remarkably, the new compound 1 exhibited cytotoxic activities against HeLa and MCF-7 cell lines with IC 50 values of 0.30 and 0.33 µM, respectively.Compound 8 was not evaluated in cytotoxicity assays for the scarcity of material.

Material and Fermentation
The strain 219807 was isolated from mangrove soil collected in Sanya, Hainan Province, China and characterized as Streptomyces sp. by 16S rRNA sequence (GenBank accession number HQ992731).A voucher specimen was deposited in the Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Wuhan University, Wuhan, China), Ministry of Education, and Wuhan University School of Pharmaceutical Sciences (http://pharmacy.whu.edu.cn/NewsDetail.asp?MaxSort=xygk&MaxUrl= about&id=450) and the China Center for Type Culture Collection (CCTCC No: M 2015276).
The strain was pre-cultured on ISP2 agar medium (0.4% glucose, 0.4% yeast extract, 1% malt extract, pH 7.0, supplemented with 2% agar) for 5-7 days.A single colony was inoculated into 50 mL of ISP2 liquid medium in 250 mL Erlenmeyer flasks on a rotatory shaker (220 rpm) at 28 ˝C for 3 days.Subsequently, 10 mL of seed culture was inoculated to 1 L Erlenmeyer flask containing 250 mL of DO fermentation medium (1% glucose, 2.5% dextrin, 2% oatmeal, 1% cottonseed flour, 0.5% fish meal, 0.2% yeast extract, 0.3% CaCO 3 , pH 6.0).Total 5 L of the fermentation broth was incubated for 8 days at 220 rpm and 28 ˝C.The highest yield of elaiophylin (4) was obtained while using 50 mL liquid culture of DO medium in 250 mL flask, observed by HPLC and calculated based on its standard curve [25].

Halichoblelide D ( 1 )
was obtained as a white needle-like crystals.Its molecular formula was determined as C 49 H 80 O 15 by the negative HRESIMS at m/z 943.5183 [M + Cl] ´(calc.for C 49 H 80 O 15 Cl 13 C-NMR and DEPT spectra illustrated characteristic signals attributable to two carbonyls at δ C 170.3, 170.2, two same groups of olefinic methines at δ C 121.2, 145.3, 132.3, 144.5, along with two hemiketal carbon at δ C 99.3, 99.4.Through detailed analysis of NMR data, the fragments of 1 resembled those of the known compound elaiophylin (4) (please see Supplementary Materials Table