New Antifungal Metabolites from the Mariana Trench Sediment-Associated Actinomycete Streptomyces sp. SY1965

New streptothiazolidine A (1), streptodiketopiperazines A (2) and B (3), and (S)-1-(3-ethylphenyl)-1,2-ethanediol (4), together with eight known compounds (5–12), were isolated from the Mariana Trench sediment-associated actinomycete Streptomyces sp. SY1965. The racemic mixtures of (±)-streptodiketopiperazine (2 and 3) and (±)-1-(3-ethylphenyl)-1,2-ethanediol (4 and 5) were separated on a chiral high-performance liquid chromatography (HPLC) column. Structures of the new compounds were elucidated by their high-resolution electrospray ionization mass spectroscopy (HRESIMS) data and extensive nuclear magnetic resonance (NMR) spectroscopic analyses. Streptothiazolidine A is a novel salicylamide analogue with a unique thiazolidine-contained side chain and its absolute configuration was established by a combination of nuclear Overhauser effect spectroscopy (NOESY) experiment, electronic circular dichroism (ECD) and 13C NMR calculations. New streptothiazolidine A (1) and streptodiketopiperazines A (2) and B (3) showed antifungal activity against Candida albicans with MIC values of 47, 42, and 42 g/mL, respectively.


Introduction
The deep-sea organisms under extreme conditions have had to make significant biochemical and physiological adaptations for survival, which result in the modification of both gene regulation and metabolic pathways to produce metabolites with unique structures and bioactivities that differ from those produced by shallow-water organisms [1][2][3]. It was reported that about 75% of deep-sea natural products have biological activity, about 40% are drug-like, and 2/3 are within Known Drug Space (KDS) [2]. The well-known example is salinosporamide A, which was isolated from a marine obligate actinomycete Salinospora tropica strain CNB-392 [4]. Salinosporamide A is a second-generation proteasome inhibitor and currently termed as marizomib under investigation for the treatment of relapsed-refractory multiple myeloma and malignant glioma [5,6]. The marine obligate Salinospora actinomycetes were found in tropical and subtropical marine sediments at depths of up to 1100 m [7,8], and most of the secondary metabolites reported to date from the genus Salinospora are novel structural molecules [9].
The Mariana Trench is the deepest known site in the Earth's oceans, reaching a depth of about 11,000 m at the Challenger Deep. Recent studies revealed that Mariana Trench sediments are enriched in microorganisms [10][11][12], however, the structures and bioactivities of their secondary metabolites are poorly known. During the course of our ongoing research program for the discovery metabolites are poorly known. During the course of our ongoing research program for the discovery of novel bioactive agents from marine microorganisms [13][14][15][16][17], an actinomycete strain SY1965 was isolated from a sediment sample collected from the Mariana Trench at depth of 11,000 m. An extract prepared from the culture of this hadal actinomycete in Gauze's liquid medium with sea salt showed antiproliferative activities against human glioma U87MG and U251 cells with an inhibition rate of over 100%. Chemical investigation on this active extract resulted in the isolation of 12 secondary metabolites (1-12), including new streptothiazolidine A (1), streptodiketopiperazines A (2) and B (3), and (S)-1-(3-ethylphenyl)-1,2-ethanediol (4) (Figure 1). In this study, we report the isolation, structure elucidation, and bioactive evaluation of these isolated marine natural products.

Results and Discussion
The Mariana-Trench-sourced strain SY1965 ( Figure S1, Supplementary Materials) was identified as Streptomyces sp. SY1965, according to its 16S rDNA sequence (1416 bp, Figure S2), which was over 99.6% match to those of several Streptomyces strains (Table S1). An extract prepared from a large-scale culture of the strain SY1965 in Gauze's liquid medium with sea salt was separated by column chromatography, followed by high performance liquid chromatography (HPLC) purification, to afford compounds 1-12.

Results and Discussion
The Mariana-Trench-sourced strain SY1965 ( Figure S1, Supplementary Materials) was identified as Streptomyces sp. SY1965, according to its 16S rDNA sequence (1416 bp, Figure S2), which was over 99.6% match to those of several Streptomyces strains (Table S1). An extract prepared from a large-scale culture of the strain SY1965 in Gauze's liquid medium with sea salt was separated by column chromatography, followed by high performance liquid chromatography (HPLC) purification, to afford compounds 1-12.

General Experimental Procedures
Ultraviolet (UV), infrared radiation (IR), optical rotation (OR), and electronic circular dichroism (ECD) spectra were measured on a METASH UV-8000 (Shanghai METASH Instruments Co. Ltd., Shanghai, China), a Nicolet TM IS TM 10 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), a RUDOLPH AutopolⅠAutomatic polarimeter, and a JASCO J-815 spectropolarimeter (JASCO Co. Tokyo, Japan), respectively. HRESIMS data were obtained on an Agilent 6230 Time of Flight Liquid Chromatography/Mass Spectrometry (TOF LC/MS) spectrometer. NMR data were acquired on a JEOL 600 spectrometer, using a standard JEOL pulse sequences for 1D and 2D (gHMQC, gHMBC, and gCOSY) NMR experiments and chemical shifts were expressed in δ (ppm) relative to All isolated compounds 1-12 were tested for their antiproliferative activity against human glioma U87MG and U251 cells by the Sulforhodamine B (SRB) assay [35]. Doxorubicin (DOX, an anticancer drug) was used as a positive control. Unfortunately, none of these tested compounds showed antiproliferative activity at a concentration of 50 µM. Obviously, the antiproliferative compounds responsible for the activity of the crude extract were lost during the process of separation.

Isolation and Taxonomic Identification of Strain SY1965
Strain SY1965 was isolated from hadal sediments MTD11000, which was collected from the Mariana Trench (11 • 20 N and 142 • 11.5 E) at depth 11,000 m on November, 2018. Briefly, the soil was dried at 55 • C for 90 min to stimulate the germination of actinomycete spores and the dried soil (1 g) was diluted with sterile water to make dilutions of 10 −1 , 10 −2 , and 10 −3 g/mL. Each dilution (200 µL) was covered on the surface of different solid media and then incubated at 28 • C for 14 days. The single pure colony of SY1965 was picked from the 10 −1 g/mL suspension in Gauze's agar medium and then transferred to another fresh Gauze's agar medium plate. After another 3 days of growth at 28 • C, the single colony (SY1965) that grew well was transferred onto Gauze's agar slants and stored at 4 • C for further study.
The 16S rDNA analysis of strain SY1965 was performed by Legenomics (Hangzhou, China) and its DNA sequence was compared to those in the GenBank database using nucleotide BLAST (Basic Local Alignment Search Tool). The 16S rDNA sequence of strain SY1965 has been deposited in GenBank (accession number: MT421944). The strain Streptomyces sp. SY1965 was preserved at the Laboratory of Institute of Marine Biology and Pharmacology, Ocean College, Zhoushan campus, Zhejiang University, Zhoushan, China.

Mass Culture of Strain SY1965
The pure colonies of SY1965 were inoculated in an Erlenmeyer flask (500 mL) containing 250 mL Gauze s liquid medium and then incubated at 28 • C for three days on a rotary shaker at 180 rpm to produce seed broth. The seed broth (10 mL) was transferred into a 500 mL Erlenmeyer flask, which contained 250 mL Gauze's liquid medium with sea salt. All flasks were placed on rotary shakers at 180 rpm for incubation at 28 • C for 14 days. A total of 50 L culture was prepared for this study.

Extraction and Isolation of Compounds 1-12
The 50 L culture of strain SY1965 was centrifuged to yield filtrate and mycelia. The mycelia were extracted with MeOH three times to give mycelium extract. The filtrate was absorbed onto a HP-20 column eluting with water and then MeOH to obtain crude filtrate extract. The mycelium and filtrate extracts were combined and then partitioned with EtOAc three times to afford a crude extract (2 g). This crude extract (2 g) was subjected to a column of ODS eluting successively with 30% MeOH, 50% MeOH, 70% MeOH, and 100% MeOH to give three fractions (Frs. A-C) based on the results of TLC and HPLC analyses. Fr. A was fractionated by Sephadex LH-20 using 40% MeOH to give subfractions A 1 -A 3 . Fr. A 1 was separated by using CXTH LC-3000 preparative HPLC (column: Fuji-C 18 CT-30, 280 × 30 mm, 10 µm; flow rate: 10 mL/min; UV detection: 210 nm) with linear gradient from 30% to 60% MeOH in 40 min, isocratic elution with 60% MeOH from 40.01 to 50 min to afford a mixture of 6 and 7 (5 mg, t R

ECD Calculation
Monte Carlo conformational searches were carried out by means of the Spartan's 10 software using Merck Molecular Force Field (MMFF). Five low-energy conformers of each model molecule of 8R,9R,11R-1, 8S,9R,11R-1, 8R,9S,11S-1, and 8S,9S,11S-1 (Tables S7, S9, S11 and S13) were selected for ECD calculations. The selected conformers were then optimized at the B3LYP/6-31+g (d, p) level in MeOH using the conductor-like polarizable continuum model (CPCM). The theoretical calculation of ECD was conducted in MeOH using Time-Dependent Density Functional Theory (TD-DFT) at the B3LYP/6-311+g (d, p) level for all selected conformers. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California, San Diego, CA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.

13 C NMR Calculations
Monte Carlo conformational searches were carried out by means of the Spartan's 10 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmann population of over 5% were chosen for NMR calculations, and then the conformers were initially optimized at B3LYP/6-31g (d, p) level in gas. Meanwhile, gauge-independent atomic orbital (GIAO) calculations of 13 C NMR chemical shifts were accomplished by density functional theory (DFT) at the mPWLPW91-SCRF (DMSO)/6-311+g (d, p) level with the PCM solvent continuum model in Gaussian 09 software. The calculated NMR data of the lowest energy conformers for 8R,9R,11R-1 and 8S,9R,11R-1 were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy. The 13 C NMR chemical shifts for TMS were calculated by the same protocol and used as reference [37]. The experimental and calculated data were analyzed by the improved probability DP4+ method for isomeric compounds. A significantly higher DP4 + probability score of 8R,9R,11R-1 and 8S,9R,11R-1 suggested the correctness of its configuration.

Culture of Human Glioma Cells
Human glioma U87MG and U251 cells were cultured in Minimum Essential Medium (MEM), Gibco and Dulbecco's Modified Eagle Medium (DMEM), Gibco with 10% FBS, respectively. All cells were incubated at 37 • C in a humidified incubator with 5% CO 2 incubator. Cells after the third generation were used for experiment.

Sulforhodamine B (SRB) Assay
The SRB assay, as described in a previous report [35], was applied to determine the antiproliferative activity of all isolated compounds 1-12 against human glioma U87MG and C251 cells. Doxorubicin (DOX) was used as a positive control.

Antimicrobial Assay
The antimicrobial activities of compounds 1-12 inhibiting the growth of MRSA, E. coli and C. albicans were also evaluated by the micro-broth dilution method, as described in previous publication [36]. Vancomycin, gentamicin, and amphotericin B were used as positive controls and DMSO was used as a negative control.