Isolation of Petrocidin A, a New Cytotoxic Cyclic Dipeptide from the Marine Sponge-Derived Bacterium Streptomyces sp. SBT348

A new cyclic dipeptide, petrocidin A (1), along with three known compounds—2,3-dihydroxybenzoic acid (2), 2,3-dihydroxybenzamide (3), and maltol (4)—were isolated from the solid culture of Streptomyces sp. SBT348. The strain Streptomyces sp. SBT348 had been prioritized in a strain collection of 64 sponge-associated actinomycetes based on its distinct metabolomic profile using liquid chromatography/high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR). The absolute configuration of all α-amino acids was determined by HPLC analysis after derivatization with Marfey’s reagent and comparison with commercially available reference amino acids. Structure elucidation was pursued in the presented study by mass spectrometry and NMR spectral data. Petrocidin A (1) and 2,3-dihydroxybenzamide (3) exhibited significant cytotoxicity towards the human promyelocytic HL-60 and the human colon adenocarcinoma HT-29 cell lines. These results demonstrated the potential of sponge-associated actinomycetes for the discovery of novel and pharmacologically active natural products.


Introduction
Marine sponges are sources for diverse and novel actinomycetes [1][2][3][4]. A multitude of secondary metabolites [5] have been recovered from marine sponge-associated actinomycetes that display diverse biological activities, such as antimicrobial [6][7][8][9], antiparasitic [10,11], immunomodulatory [12], antichlamydial [13], antioxidant [14], and anticancer [15,16] activities. The conventional natural products isolation scheme has however frequently been challenged owing to the repeated isolation of known compounds, the failure to isolate trace compounds and sometimes the loss of activity during fractionation and compound purification [17]. The lack of detailed chemical information prior to isolation is another draw-back of conventional natural product discovery. By comparison, the metabolomics approach comes with the significant advantage that different active compounds can be determined in crude extracts without comprehensive fractionation or prior isolation of the individual compound while still offering structural information [18][19][20][21]. Our previous study using individual compound while still offering structural information [18][19][20][21]. Our previous study using metabolomics and dereplication tools revealed a chemically distinct strain Streptomyces sp. SBT348 by PCA analysis [22]. In the present study, we report on the isolation and structure elucidation of a new cyclic dipeptide, petrocidin A (1); three known compounds 2,3-dihydroxybenzoic acid (2), 2,3-dihydroxybenzamide (3), and maltol (4) from strain Streptomyces sp. SBT348; as well as the determination of their anti-proliferative properties against HL-60, HT29, and MCF-7 cancer cell lines.

Isolation and Purification of Strain Streptomyces sp. SBT348
Streptomyces sp. SBT348 (GenBank accession No. KP238417) was cultivated from the Mediterranean sponge Petrosia ficiformis that was collected from Milos, Greece [22]. Our study using metabolomics and dereplication tools prioritized the strain Streptomyces sp. SBT348 based on its chemical uniqueness by Principal Component Analysis (PCA) (Figure 1). The ethyl acetate extract of the solid culture of Streptomyces sp. SBT348 was fractionated by preparative HPLC to afford a new cyclic dipeptide (1), and three known compounds-2,3-dihydroxybenzoic acid (2) [23], 2,3dihydroxybenzamide (3) [24], and maltol (4) [25]. The structures of compounds 2, 3, and 4 were determined based on the MS and NMR elucidation, as well as comparison to the literature and Integrated Spectral Database System of Organic Compounds developed by AIST (National Institute of Advanced Industrial Science and Technology). The structure elucidation of the new cyclic dipeptide compound 1 was discussed as follows.

Structure Elucidation
Compound 1 (1.8 mg, yield 0.49%) was purified as a colorless solid. The following NMR spectral data were acquired using a 600 MHz instrument: 1 H, 13 C, 13

Structure Elucidation
Compound 1 (1.8 mg, yield 0.49%) was purified as a colorless solid. The following NMR spectral data were acquired using a 600 MHz instrument: 1 H, 13 C, 13 13 C, 3 C-DEPT135 and HSQC spectra displayed six aromatic carbons at δ C 114.7, 119.6, 121.3, 121.9, 147.0, and 151.7 ppm; three ester/amide-type carbonyls at δ C 168.3, 172.8, and 174.2 ppm; three methine carbons at δ C 60.3, 54.6, and 25.8 ppm; four methylene carbons at δ C 23.7, 29.1, 39.4, and 46.4 ppm; and two methyl carbons at δ C 22.2 and 23.2 ppm. The three aromatic protons at δ H 6.98, 6.71, and 7.34 ppm were assigned to H-4, H-5, and H-6, respectively by COSY spectrum and indicated an adjacent three-substitution aromatic system as part of the structure. The deshielded carbon resonances at δ C 151.7 and 147.0 ppm suggested two hydroxyl substitutions on the aromatic ring, and were assigned to C-2 and C-3 respectively, with the correlations observed amongst H-4, H-5, and H-6 in the HMBC spectrum ( Figure 2). The other aromatic carbon resonances at δ C 114.7, 121.3, 119.6, 121.9 ppm were attributed to C-1, C-4, C-5, C-6, respectively according to the cross-peaks observed in the HSQC and HMBC spectra. The strong correlation observed in the HMBC spectrum between H-6 and one of the ester/amide-type carbonyls at δ C 174.2 ppm has assigned δ C 174.2 to C-7 ( Figure 2). The HSQC and HMBC spectra also exhibited standard resonance signals for amino acids by the observation of two α-methines signals at δ H 4.26 (1H, t, J = 8.5, 16.1 Hz), 4.13 (1H, t, J = 5.6, 11.4 Hz) with their connected deshielded carbons at δ C 60.3 and 54.6 ppm, respectively, as well as HMBC cross-peaks between the α-protons and ester/amide-type carbonyls at δ C 172.8 and 168.9 ppm ( Figure 2). Analysis of the COSY, HSQC, and HMBC data, as well as degrees of unsaturation assigned one ornithine and one leucine moiety presented in a cyclic nature. The connection between the aromatic part and the cyclic peptide moiety was confirmed by the correlation displayed in HMBC (using DMSO-d 6 as solvent) between Orn β-H and C-7 ( Figure 2). The proton and carbon resonances were also compared to the human metabolites database (HMDB) with ornithine and leucine individually, as well as 2,3-dihydroxybenzamide-scaffold with compound 2 and literature [24], and they showed consistent results. The absolute configuration of leucine and ornithine was determined by acid hydrolysis followed by Marfey's derivatization and comparison to their analogues of the authentic amino acids by HPLC analysis. The Marfey's analysis of compound 1 revealed the absolute configuration of leucine to be L and ornithine to be D-configuration. Compound 1 was identified as 2,3-dihydroxy-N-((3S,6R)-3-isobutyl-2,5-dioxo-1,4-diazonan-6-yl) benzamide, a new cyclic dipeptide, and given the name petrocidin A. Compounds 2, 3, and 4 were isolated as colorless solid from fraction 1, 2, and 5 respectively. The molecular formula was established as C 7 H 6 O 4 (found at EI m/z 154.0, calcd. 154.0266) for compound 2, C 7 H 7 NO 3 (found at EI m/z 153.0, calcd. 153.0426) for compound 3, and C 6 H 6 O 3 (found at EI m/z 126.0, calcd. 126.0317) for compound 4, respectively. An exact mass search in the Database of Natural Products (2015) and comparison of the spectral data with the literature determined compound 2 as 2,3-dihydroxybenzoic acid [26], compound 3 as 2,3-dihydroxybenzamide [24], and compound 4 as Maltol [27]. and six methyl protons at δH 0.96 (3H, d, J = 3.7 Hz) and 0.95 (3H, d, J = 3.7 Hz) ppm. 13 C, 3  , and H-6, respectively by COSY spectrum and indicated an adjacent threesubstitution aromatic system as part of the structure. The deshielded carbon resonances at δC 151.7 and 147.0 ppm suggested two hydroxyl substitutions on the aromatic ring, and were assigned to C-2 and C-3 respectively, with the correlations observed amongst H-4, H-5, and H-6 in the HMBC spectrum ( Figure 2). The other aromatic carbon resonances at δC 114.7, 121.3, 119.6, 121.9 ppm were attributed to C-1, C-4, C-5, C-6, respectively according to the cross-peaks observed in the HSQC and HMBC spectra. The strong correlation observed in the HMBC spectrum between H-6 and one of the ester/amide-type carbonyls at δC 174.2 ppm has assigned δC 174.2 to C-7 ( Figure 2). The HSQC and HMBC spectra also exhibited standard resonance signals for amino acids by the observation of two α-methines signals at δH 4.26 (1H, t, J = 8.5, 16.1 Hz), 4.13 (1H, t, J = 5.6, 11.4 Hz) with their connected deshielded carbons at δC 60.3 and 54.6 ppm, respectively, as well as HMBC cross-peaks between the α-protons and ester/amide-type carbonyls at δC 172.8 and 168.9 ppm (Figure 2). Analysis of the COSY, HSQC, and HMBC data, as well as degrees of unsaturation assigned one ornithine and one leucine moiety presented in a cyclic nature. The connection between the aromatic part and the cyclic peptide moiety was confirmed by the correlation displayed in HMBC (using DMSO-d6 as solvent) between Orn β-H and C-7 ( Figure 2). The proton and carbon resonances were also compared to the human metabolites database (HMDB) with ornithine and leucine individually, as well as 2,3dihydroxybenzamide-scaffold with compound 2 and literature [24], and they showed consistent results. The absolute configuration of leucine and ornithine was determined by acid hydrolysis followed by Marfey's derivatization and comparison to their analogues of the authentic amino acids by HPLC analysis. The Marfey's analysis of compound 1 revealed the absolute configuration of leucine to be L and ornithine to be D-configuration. Compound 1 was identified as 2,3-dihydroxy-N-((3S,6R)-3-isobutyl-2,5-dioxo-1,4-diazonan-6-yl) benzamide, a new cyclic dipeptide, and given the name petrocidin A. Compounds 2, 3, and 4 were isolated as colorless solid from fraction 1, 2, and 5 respectively. The molecular formula was established as C7H6O4 (found at EI m/z 154.0, calcd. 154.0266) for compound 2, C7H7NO3 (found at EI m/z 153.0, calcd. 153.0426) for compound 3, and C6H6O3 (found at EI m/z 126.0, calcd. 126.0317) for compound 4, respectively. An exact mass search in the Database of Natural Products (2015) and comparison of the spectral data with the literature determined compound 2 as 2,3-dihydroxybenzoic acid [26], compound 3 as 2,3-dihydroxybenzamide [24], and compound 4 as Maltol [27].
Microsomal prostaglandin E 2 synthase-1 (mPGES-1) has been reported to be over-expressed in a group of tumor cells-such as HL-60, HT-29, and MCF-7 cell lines-but not in normal tissues [35,36]. The moiety of 2,3-dihydroxybenzamide was proposed as an active scaffold in the search for new mPGES-1 inhibitors used in anti-inflammatory and anticancer process [37]. The anti-proliferative properties of the four compounds were evaluated against the three cancer cell lines including the HL-60, HT-29, and MCF-7 cell lines. Petrocidin A (1) displayed significant cytotoxic effects towards HL-60 and HT-29 cells with the IC 50 values of 3.9 and 5.3 µg/mL, respectively. While, 2,3-Dihydroxybenzamide (3) exhibited potent cytotoxicity towards the same two cell lines with the IC 50 values of 5.5, and 3.8 µg/mL, respectively. 2,3-Dihydroxybenzoic acid (2) and maltol (4) did not exhibit any significant cytotoxicity at the examined concentrations. These results suggested that the new cyclic dipeptide petrocidin A (1) and 2,3-dihydroxybenzamide (3) which is a part of the peptidic structure exhibiting anti-proliferative effects towards HL-60 and HT29 cancer cell lines which might be as mPGES-1 inhibitors.

Large-Scale Fermentation, Extraction, and Isolation
Three hundred ISP2 agar plates (square 120 × 120 mm), each inoculated with 100 µL of five days liquid culture of Streptomyces sp. SBT348 respectively were incubated at 30 • C for seven days. The agar media with bacterial biomass were scalped into small pieces and transferred to 1 L Erlenmeyer flasks. Five hundred mL of ethyl acetate/flask were added to submerge the agar pieces and macerated the medium culture under shaking at 175 rpm for overnight. The macerations were subsequently filtered by gravity using filter paper (A. Hartenstein, Würzburg, Germany). The filtrates were combined and evaporated by under vacuum (Büchi, Essen, Germany). Three-hundred and seventy mg of the dried crude EtOAc extract obtained from the solid culture of strain Streptomyces sp. SBT348 was fractionated by semi-preparative HPLC (Agilent, Waldbronn, Germany) using H 2 O/ACN (95%:5%) initially for 5 min, then by a linear gradient to 100% ACN within 40 min, which was followed by an isocratic condition of 100% ACN for a further 5 min on the Prep C18 column (5 µm, 10 × 250 mm, (Waters XBridge, Eschborn, Germany), with a flow rate of 3.0 mL/min to yield five fractions. Compounds 1-4 were purified from peaks rich fraction Nr. 3 by semi-preparative HPLC using Onyx Monolithic semi-prep RP-C18 column (5 µm, 10 × 100 mm, Phenomenex, Aschaffenburg, Germany).

LC-MS Analysis
Accurate electrospray ionization mass spectra (ESI) were obtained by a Synapt G2 HDMS qTOF-Mass Spectrometer (Waters, Eschborn, Germany). The ESI was operated in the positive and negative ionization modes. The capillary voltage was set to 0.8 kV and nitrogen (at 350 • C, the flow rate of 800 L/h) was used as desolvation gas. The molecular ion was determined by quadrupole in a wide-band RF mode, and data was acquired over the mass range of 50-1200 Da. Product ion scan was optimized for different analyses using different collision voltage from 10 to 25 eV. MassLynx (version 4.1, Waters, Bremen, Germany) was utilized to acquire and process mass spectrum.

Marfey's Analysis
The absolute configuration of α-H that presented in compound 4 was performed by Marfey's derivatization and compared to the purchased amino acid with D and L configurations (Sigma, Darmstadt, Germany) by HPLC. Compound 4 (0.8 mg) was initially hydrolyzed with 6 M HCl Mar. Drugs 2017, 15, 383 6 of 9 (2 mL) in the water bath at 100 • C for 24 h. The hydrolysate was cooled to room temperature, dried using vacuum evaporator, and finally dissolved in 100 µL of water. The Marfey's derivatization was carried out by adding 100 µL of 1% Marfey's reagent (1-Fluoro-2,4-dinitrophenyl-5-L-alanine amid) dissolved in acetone and 20 µL of 1 M NaHCO 3 (H 2 O) to 50 µL of the hydrolysate of compound 4, as well as 50 mM standard amino acid (D-Leu, L-Leu, D-Orn, and L-Orn) respectively, and incubated at 40 • C for 1 h with frequent shaking. The reaction was stopped by adding 10 µL of 2 M HCl after cooling. The Marfey's derivatization products were finally dried and prepared in MeOH for further HPLC analysis. The HPLC chromatography was carried out on Gemini-NX RP-C18 column (Phenomenex, Aschaffenburg, Germany by eluting with H 2 O/CH 3 CN (95%:5%) for the first 5 min, linearly gradient to 100% CH 3 CN within 25 min, and held at 100% CH 3 CN for a further 5 min with a flow rate at 1 mL/min and UV detection at 340 nm. The configuration was eventually determined with the observation of the same retention times compared to the purchased standard enantiomeric amino acids (Bhushan and Bruckner, 2004; Kochhar and Christen, 1989; Marfey, 1984).
3.6. MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) Assay Cell proliferation was evaluated in cell lines by the MTT assay in triplicates. Briefly, cells were plated in a 96-well microtiter plate at a density of 1 × 10 4 cells per well in a final volume of 100 µL of culture medium. These cells were treated for 24 h with tested compound at 37 • C with 5% CO 2 . After treatment, the cells were immediately incubated with 10 µL MTT (5.0 mg/mL) for 4 h at 37 • C. The cells were lysed in 100 µL of lysis buffer (isopropanol, conc. HCl and Triton X-100) for 10 min at room temperature and 300 rpm/min shaking. The enzymatic reduction of MTT to formazan crystals that dissolved in DMSO was quantified by photometry at 570 nm. Dose-response curves were generated and the IC 50 values were defined as the concentration of compound required to inhibit cell proliferation by 50%. 5-Flurouracil was used as a positive control.