Unique Cyclized Thiolopyrrolones from the Marine-Derived Streptomyces sp. BTBU20218885

Two new cyclized thiolopyrrolone derivatives, namely, thiolopyrrolone A (1) and 2,2-dioxidothiolutin (2), together with the kn own compound, thiolutin (3) were identified from a marine-derived Streptomyces sp. BTBU20218885, which was isolated from a mud sample collected from the coastal region of Xiamen, China. Their chemical structures were determined using spectroscopic data, including HRESIMS, 1D and 2D NMR techniques. 1 possessed a unique unsymmetrical sulfur-containing thiolopyrrolone structure. All the compounds were tested for bioactivities against Staphylococcus aureus, Escherichia coli, Bacille Calmette–Guérin (BCG), Mycobacterium tuberculosis, and Candida albicans. 1 displayed antibacterial activities against BCG, M. tuberculosis, and S. aureus with minimum inhibitory concentration (MIC) values of 10, 10, and 100 μg/mL, respectively. Thiolutin (3) showed antibacterial activities against E. coli, BCG, M. tuberculosis, and S. aureus with MIC values of 6.25, 0.3125, 0.625, and 3.125 μg/mL, respectively.


Introduction
Infectious diseases caused by infectious microorganisms continue to threaten human health. Moreover, the development of drug resistance by Candida albicans, Staphylococcus aureus, Escherichia coli, and Mycobacterium tuberculosi is becoming more and more serious in hospitals and the community [1][2][3][4]. There is an urgent need to develop new drugs to fight against these pathogens.
In the course of our screening of antibacterial secondary metabolites from marinederived actinomycetes [21][22][23][24], the EtOAc extract of Streptomyces sp. BTBU20218885, isolated from a mud sample collected from the coastal area of Xiamen, Fujian Province, China, showed antibacterial activity against Bacille Calmette-Guérin (BCG), the live attenuated vaccine form of Mycobacterium bovis, with minimum inhibitory concentration (MIC) of 20 µg/mL. A chemical investigation of this Streptomyces strain resulted in the isolation of two new cyclized thiolopyrrolone derivatives, namely, thiolopyrrolone A (1) and 2,2dioxido thiolutin (2), together with the known compound, thiolutin (3) (Figure 1). Details of fermentation, isolation, structural elucidation, and antibacterial activities are reported here.

General Experimental Procedures
NMR spectra were obtained on a Bruker Avance 500 spectrometer with residual solvent peaks as references (DMSO-d 6 : δ H 2.50, δ C 39.52). High-resolution ESIMS measurements were obtained on an Accurate-Mass-Q-TOF LC/MS 6520 instrument (Santa Clara, CA, USA) in the positive ion mode. HPLC was performed using an Agilent 1200 Series separation module equipped with an Agilent 1200 Series diode array, Agilent 1200 Series fraction collector, and Agilent ZORBAX SB-C18 column (250 × 9.4 mm, 5 µm).

Microbial Material, Fermentation, Extraction, and Purification
Strain Streptomyces sp. BTBU20218885 was isolated from a mud sample collected from the intertidal zone, Xiamen, China, and grown on an ISP2 (yeast extract 0.4%, malt extract 1%, dextrose 0.4%, agar 2%; pH 7.2) agar plate at 28 • C. Colony characteristics of BTBU20218885 are shown in Figure S17. The genomic DNA of BTBU20218885 was extracted using a TINAamp Bacteria DNA Kit. PCR amplification of 16S rDNA was carried out by using universal primers (27f:5 -GAGAGTTTGATCCTGGCTCAG-3 ; 1492r: 5 -CTACGGCTACCTTGTTACGA-3 ). PCR amplification of the 16S rDNA was performed on TaKaRa PCR Thermal Cycler with the initial denaturation at 94 • C for 5 min, 30 cycles of denaturation (94 • C, 1 min), annealing (55 • C, 1 min), and elongation (72 • C, 1 min 15 s), and a final elongation at 72 • C for 10 min, in a 25 µL system (0.4 µL 20 µM of each primer, 2.5 µL 10× buffer, 2.5 µL 2.5 nM dNTP, 2 U rTap polymerase, and 1 µL DNA template). BTBU20218885 was identified as Streptomyces sp. by comparing the 16S rDNA sequence with the GenBank database using the BLAST program. A neighbor-joining (NJ) tree ( Figure S18) was constructed using the software package Mega version 5 [26]. The strain was assigned the accession number BTBU20218885 in the culture collection at Beijing Technology and Business University, Beijing. The strain BTBU20218885 was inoculated on an ISP2 agar plate and cultured for 7 days. A 250 mL Erlenmeyer flask containing 40 mL of ISP2 medium was inoculated with BTBU20218885 and incubated at 28 • C (160 rpm) for 36 h. Aliquots (9 mL) of the seed cultures were aseptically transferred to 20 × 1 L Erlenmeyer flasks, each containing 300 mL of MPG media (glucose 1.0%, millet meal 2.0%, cotton seed gluten meal 2.0%, and MOPS 2.0%; pH 7.0), and the flasks were incubated at 28 • C, 160 rpm for 7 days. The culture broths were combined and centrifuged to yield a supernatant and a mycelial cake. The supernatant was extracted by equal volume of ethyl acetate (EtOAc, ×3 times), and the combined EtOAc extracts were evaporated to dryness in vacuo to give a dark residue. The residue was sequentially triturated with hexane, CH 2 Cl 2 , and MeOH to afford, after concentration in vacuo, hexane, CH 2 Cl 2 , and MeOH soluble fractions and precipitate. The precipitate was further purified by HPLC (Agilent ZORBAX SB-C18, 250 × 9.4 mm, 5 µm column, 3.0 mL/min, elution with 30% to 100% acetonitrile/H 2 O (0-20 min) to yield 1 (3.5 mg), 3 (2.6 mg), and 2 (13.2 mg).

Biological Activity
Compounds 1-3 were evaluated for their antimicrobial activities in 96-well plates according to the Antimicrobial Susceptibility Testing Standards outlined by the Clinical and Laboratory Standards Institute Document M07-A7 (CLSI) and our previous report [27][28][29]. The MIC was defined as the minimum concentration of the compound that prevented visible growth of the microbes.

Computational Methods
A random conformational search of starting geometries in Discovery studio 4.0 was used to produce low-energy conformers within a 10 kcal/mol energy, which were subsequently optimized using the DFT method at mPW1PW91/6-31g(2d,p) level with GAUS-SIAN 09 [30]. The optimized conformers were further checked by frequency calculation at the same level of theory, and resulted in no imaginary frequencies. The time-dependent density functional theory (TDDFT) calculations of their low-energy conformations within 0-2.5 kcal/mol were performed to simulate their UV-vis spectra at the same level. Similarly, their 13 C NMR calculations were also carried out by GIAO method at the same level [31]. Solvent effect of dimethylsulfoxide was taken into account in the above calculations by using the polarizable continuum model (PCM).
Their theoretical UV-vis spectra based on Boltzmann statistics were generated in the program SpecDis 1.63 [32] by applying Gaussian band shape with a 0.40 eV exponential half-width from dipole-length rotational strengths. Statistical parameters were used to quantify the agreement between experimental and calculated data, including the correlation coefficient (R 2 ) between experimental and calculated 13 C NMR spectroscopic data with a linear regression, the mean absolute error (MAE), and the maximum error (MaxErr) [33].
The correlation coefficient (R 2 ) was determined from a plot of δ calc (x axis) against δ exp (y axis) for each particular compound. The mean absolute error (MAE) was defined as 1 n n ∑ i=1 δ calc, i − δ exp, i . The maximum error (MaxErr) was defined as max|δ calc − δ exp |.

Conclusions
In summary, chemical studies on the marine-derived Streptomyces sp. BTBU20218885 resulted in the characterization of three cyclized thiolopyrrolones, including a unique unsymmetrical thiolopyrrolone (1), 2,2-dioxidothiolutin (2), and the previously reported thiolutin (3). Dithiolopyrrolones are a class of structurally intriguing natural products with broad antibacterial spectrum [34]. Most of the analogues are characterized by a unique bicyclic pyrrolinonodithiole, with the differences in the substitution groups on N-4 and N-7 positions of the holothin core [35,36]; however, thiolopyrrolone A is the first sample of analogues with a macrocyclic skeleton. Compound 1 exhibited antibacterial activities against BCG, M. tuberculosis, and S. aureus with MIC values of 10, 10, and 100 µg/mL, respectively. Thiolutin (3) displayed potential antibacterial activities against E. coli, BCG, M. tuberculosis, and S. aureus with MIC values of 6.25, 0.3125, 0.625, and 3.125 µg/mL, respectively.

Conflicts of Interest:
The authors declare no conflict of interest.