Rare Polyene-polyol Macrolides from Mangrove-derived Streptomyces sp. ZQ4BG

Bioactive natural products from mangrove-derived actinomycetes are important sources for discovery of drug lead compounds. In this study, an extract prepared from culture of an actinomycete Streptomyces sp. ZQ4BG isolated from mangrove soils was found to have activity in inhibiting proliferation of glioma cells. Large culture of this mangrove actinomycete in Gause’s liquid medium resulted in isolation of seven novel polyene-polyol macrolides, named as flavofungins III–IX (3–9), together with known flavofungins I (1) and II (2) and spectinabilin (10). Structures of these isolated compounds were elucidated by extensive NMR analyses and HRESIMS data. The stereochemical assignments were achieved by a combination of NOE information, universal NMR database, and chemical reactions including preparation of acetonide derivatives and Mosher esters. Flavofungins IV–VIII (4–8) are rare 32-membered polyene-polyol macrolides with a tetrahydrofuran ring, while flavofungin IX (9) represents the first example of this type of macrolide with a unique oxepane ring. Flavofungins I (1) and II (2) and spectinabilin (10) showed anti-glioma and antifungal activities.


Results and Discussion
An actinomycete strain ZQ4BG which produced rich polyene-polyol macrolides was isolated from mangrove soils and assigned as Streptomyces sp. ZQ4BG based on the result of its 16S rDNA sequence. Culture (total 60 L) of this isolated actinomycete in Gause's liquid medium resulted in isolation of compounds 1-10 ( Fig. 1).
Compounds 1, 2, and 10 were proved to be known compounds of flavofungins I (1) and II (2) 30,31 and spectinabilin (10) [32][33][34] based on their HRESIMS and NMR data (Tables 1 and 2) as well as a comparison to the data of references. Flavofungins I (1) and II (2) were 32-membered polyene-polyol macrolides with a conjugated chain of five unsubstituted double bonds connected to a polyol fragment with eight hydroxy groups alternated. Both  (2) were reported to have antifungal and antiviral activities 19 , while spectinabilin (10) was a nitro-substituted polyketide with activity to block the binding of androgen to the ligand-binding domain of androgen receptor 34 .
The NMR spectra of 9 showed 36 carbons for a carbonyl, ten olefinic methines, 11 oxymethines, three methines, seven methenes, and four methyls, which were same to those of 4, 6, and 7. Extensive NMR analyses confirmed that their structural differences were the tetrahydrofuran (THF) ring in 4, 6, and 7 replaced by an oxepane ring in 9. Treatment of 9 with 2,2-dimethoxypropane afforded triacetonide 9a and tetraacetonide 9b. Interpretation of the 13 C NMR chemical shifts (Table S3) Table S6). Unfortunately, the results from Δδ S-R values failed to assignment the configuration at C-13, probably because the two groups of Mosher agent were too close. In the NOESY spectrum of 9a (Fig. 4D) (Table S5) suggested an α-orientation for H-10 and H-15 and an β-orientation for H-11, H-13, and Me, -14. Therefore, the configurations of C-10, C-13, C-14, and C-15 were assigned as 10S, 13S, 14R, and 15R. Based on the foregoing evidences, the structure of 9 was determined as novel polyene-polyol macrolide, named as flavofungin IX.
Compound 3 gave a [M + Na] + ion at m/z 707.3972, correspondence to a molecular formula C 36 H 60 O 12 . The general features of the NMR spectra of 3 were very similar to those of 1, indicating they were structurally related compounds. The main differences were found at the positions of C-10 and C-11, where two olefinic methines  Table 3. 1 H-NMR data of compounds 5-9 (500 MHz, in DMSO-d 6 , J = Hz) a The data with the same labels in each column could not be individually assigned. of 1 were replaced by two oxymethines in 3. The UV absorption maximum at 330 nm for 3 (365 nm for 1) also supported a smaller conjugated system in 3. The configurational assignments of 11S, 13S, 14R, 15R, 17R, 19R, 21S, 23S, 25R, 27R, 30S, and 31S in 3 were achieved using the same protocol as for compounds 4-9, including 13 C Universal NMR database (Fig. 2), 13 C NMR chemical shifts (Table S3) (Table 2) in 3, suggesting 10R conformation for C-10. Therefore, the structure of 3 was assigned as a new polyene-polyol macrolide, named as flavofungin III. All isolated compounds (1-10) were evaluated by sulforhodamine B (SRB) assay for their activity in inhibiting proliferation of glioma cells U251, U87MG, SHG44, and C6. Doxorubicin (DOX) was used as a positive control. The results ( Supplementary Information, Table S2) indicated that flavofungins I (1) and II (2) and spectinabilin   (3)(4)(5)(6)(7)(8)(9) were inactive. Therefore, flavofungin II (2) and spectinabilin (10) are the main components responsible for the anti-glioma activity in the crude extract. The cytotoxicity (CC 50 ) of the two active compounds 2 and 10 towards normal human astrocyte (HA) and human foreskin fibroblast (HFF-1) cells were also assayed. The results (Table S2) indicated both compounds showed a higher selectivity index (CC 50 /IC 50 ) for HA with 4.6-16.5 for 2 and 3.4-13.3 for 10, when compared to the selectivity index for HFF-1 with 1.8-6.4 for 2 and 1.3-5.1 for 10.
Compounds (1-10) were also tested for their antimicrobial activity against methicillin-resistant Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, and Candida albicans by micro broth dilution method. Flavofungins I (1) and II (2) and spectinabilin (10) showed activity in suppressing the growth of C. albicans with MIC value of 12.5 μg/mL. The control drug amphotericin B had antifungal activity with MIC value of 3.125 μg/mL.

General Experimental Procedures. This section was supplied as online Supplementary Information.
Strain Isolation. Strains were isolated from a sample of mangrove soils, which were collected from the Qi'ao Mangrove Forest in Zhuhai City, Guangdong, China, in October, 2013. Briefly, the dried soils (1.0 g) were diluted with sterile water to make 0.001 g/mL suspension and 200 μL suspension were covered on the surface of Gause's solid medium and then incubated at 28 °C for 10 days. The single colony was picked with sterile needles and transferred to a Gause's-agar plate. After another 10 days of growth at 28 °C, the single colony that grew well was transferred onto Gause's agar slants and stored at 4 °C until use. A total of ten strains were obtained from this mangrove sample in this study.

Preparation of Crude Extract for Bioactive Assay.
Each isolated strain was cultured in Gause's solid medium with a small Petri dish (90 mm, 25 mL medium) at room temperature for 10 days. The solid culture was cut into small pieces (about 0.6 × 0.6 cm) and then percolated with MeOH three times (50 mL, each). The combined MeOH solution was dried under reduced pressure to give crude extract. This crude extract was redissolved in DMSO to prepare crude sample (1.0 mg/mL). Sulforhodamine B (SRB) assay was used to evaluate the activity of the crude sample against the proliferation of glioma U87MG and U251 cells. The crude extract prepared from the culture of strain ZQ4BG was found to be the most active and the strain ZQ4BG was thus selected for this study.
Taxonomic identity of Streptomyces sp. ZQ4BG. The 16S rDNA sequence analysis of strain ZQ4BG was conducted by Majorbio (Shanghai, China) and its DNA sequence using BLAST (nucleotide sequence comparison) was compared to the GenBank database. The 16S rDNA sequence of strain ZQ4BG has been deposited in GenBank (accession number: KX601187). The voucher strain of Streptomyces sp. ZQ4BG was preserved at the Laboratory of Institute of Marine Biology, Ocean College, Zhejiang University, China.
Large culture of strain Streptomyces sp. ZQ4BG. Colonies of the strain ZQ4BG growing on Gause's-agar slants were inoculated into a 500 mL Erlenmeyer flask containing 200 mL of Gause's liquid medium and then incubated at 28 °C for 5 days on a rotary shaker (180 rpm) to produce seed broth. The seed broth (5 mL) was inoculated into a 500 mL Erlenmeyer flask, which contains 200 mL of Gause's liquid medium. All flasks were incubated at 28 °C for 12 days on a rotary shaker (180 rpm). A total of 60 L cultures was prepared for this study.
Extraction and Isolation of compounds 1-10. The 60 L cultures were filtered to give filtrate and mycelia. The filtrate was applied to a column of Diaion HP-20 eluting with water and then 100% MeOH. The MeOH elution was concentrated in vacuo to give Part A. The mycelia were extracted with MeOH five times and the combined MeOH solution was concentrated in vacuo to afford Part B. The combination of Part A and Part B was suspended in water and then partitioned by EtOAc three times to afford a crude extract (30.0 g) after removal of the solvent EtOAc. This crude extract was fractionated on a brown column of ODS eluting successively with 70% MeOH, 80% MeOH, 90% MeOH, and 100% MeOH to give fractions A-J based on the results of TLC and LC/ MS analyses. Fraction H was separated by HPLC using an Agilent Zorbax SB-C 18 column (mobile phase: MeOH/ H 2 O, 85/15; flow rate: 1.0 mL/min) to give compounds 1 (33 mg, t R 24.3 min) and 2 (31 mg, t R 28.8 min). By using the same column and flow rate for 1 and 2 but different mobile phase, 3 (
Antimicrobial assay. The antimicrobial activity of the isolated compounds against growth of methicillin-resistant Staphylococcus aureus ATCC 43300, Escherichia coli ATCC 25922, and Candida albicans was assayed by the micro broth dilution method as described in previous study 13 . Gentamicin (an antibiotic against both Gram-positive and Gram-negative bacteria) and amphotericin B (an antifungal drug) were used as positive controls.