New Chlorinated 2,5-Diketopiperazines from Marine-Derived Bacteria Isolated from Sediments of the Eastern Mediterranean Sea.

From the organic extracts of five bacterial strains isolated from marine sediments collected in the East Mediterranean Sea, three new (15, 16, 31) and twenty-nine previously reported (1–14, 17–30, 32) metabolites bearing the 2,5-diketopiperazine skeleton were isolated. The structures of the chlorinated compounds 15, 16, and 31 were elucidated by extensive analysis of their spectroscopic data (NMR, MS, UV, IR). Compounds 15 and 16 were evaluated for their antifungal activity against Candida albicans and Aspergillus niger but were proven inactive. The relevant literature is supplemented with complete NMR assignments and revisions for the 29 previously reported compounds.


Introduction
2,5-Diketopiperazines (DKPs), also termed as cyclodipeptides, 2,5-dioxopiperazines, or dipeptide anhydrides, are the smallest possible cyclic peptides and, therefore, are among the most common peptide derivatives found in nature. They derive from the condensation of two α-amino acids forming a bis-lactam. Although they are relatively simple and low molecular weight compounds, they can be highly substituted, resulting in complex structures [1,2]. DKPs have been reported, so far, from a variety of sources, including microorganisms (bacteria and fungi), as well as higher organisms (algae, lichens, plants, marine sponges, gorgonians, tunicates, and mammals) [3,4]. They are also found in food and beverages, lending them a bitter taste [5]. The origin of DKPs has been questioned and it has been proposed that they might even be chemical degradation products. However, sterile media do not contain DKPs [6-9] and specific bacterial genes encode their biosynthesis [10][11][12].
DKPs have been neglected for many years, but they have recently attracted attention due to their chemical diversity and remarkable bioactivity. They exhibit a wide range of biological activities, such as cytotoxic, antibacterial, antifungal, antiparasitic, insecticidal, antiviral, antiprion, antifouling, antioxidant, anti-inflammatory, antihyperglycemic, and neuroprotective; thus, making them promising drug candidates. Moreover, they are involved in quorum sensing and ion-transport, and exhibit high binding affinity to a large number of receptors [4,[13][14][15][16][17].
The DKP chiral scaffold is attractive for drug design due to its simplicity, high stability (resistance to proteolysis), conformational rigidity, and remarkable structural diversity [18]. DKPs are readily accessible by chemical synthesis, constituting an excellent model for theoretical studies and an are readily accessible by chemical synthesis, constituting an excellent model for theoretical studies and an important pharmacophore in medicinal chemistry [4,13,19]. Moreover, they are employed as starting materials for the synthesis of many natural products, such as alkaloids [18].
Three drugs based on this scaffold have recently entered the market, namely tadalafil, as a phosphodiesterase-5 inhibitor for the treatment of erectile dysfunction [20], retosiban, as an oxytocin antagonist for the treatment of preterm labor [21], and epelsiban, as an oxytocin antagonist for the treatment of premature ejaculation in men [22]. Additionally, it has been shown that their presence in culture broths fermented with lactic acid bacteria (LAB) can greatly contribute to an environmentally friendly, safe, and ecological approach for food and feed preservation [23].
In the framework of our investigations towards the isolation of new bioactive secondary metabolites from marine microorganisms, a large number of bacterial strains have been isolated from marine sediments collected from the East Mediterranean basin, a relatively unexplored marine ecosystem regarding the chemistry of its microbiota. The preliminary screening of the chemical profiles of extracts obtained from small-scale liquid cultures of a large number of marine-derived bacterial strains from our microbank with LC-DAD-MS and NMR led to the selection of five strains for further chemical investigation. Extraction of large-scale cultures of the selected bacterial strains and fractionation of the obtained organic extracts allowed for the isolation of 32 DKPs (Figures 1 and  2), among which three chlorinated analogues (15,16, and 31) were identified as new natural products. Herein, we report the isolation and structure elucidation of metabolites 15, 16, and 31 and the evaluation of the antifungal activities of 15 and 16. Additionally, since several inconsistencies in the published NMR data of DKP structures are frequently observed, in conjunction with the fact that NMR data is incompletely reported for several of these, leading to confusion, complete assignment of the 1 H and 13 C NMR chemical shifts of the known metabolites 1-14, 17-30, and 32 is also presented.
Compound 15, obtained as white solid, displayed the molecular formula C14H15N2O3Cl, as deduced from high-resolution electrospray ionization mass spectrometry (HRESIMS) measurements where two isotopic sodium adduct ion peaks were observed at m/z 317.0661 and 319.0629 with a ratio of 3:1, characteristic for the presence of one chlorine atom in the molecule. The HSQC and HMBC experiments revealed 14 carbon signals, which corresponded to five non-protonated carbon Chemical structures of compounds 18-32 isolated from various marine-derived bacterial strains.
Compound 15, obtained as white solid, displayed the molecular formula C 14 H 15 N 2 O 3 Cl, as deduced from high-resolution electrospray ionization mass spectrometry (HRESIMS) measurements where two isotopic sodium adduct ion peaks were observed at m/z 317.0661 and 319.0629 with a ratio of 3:1, characteristic for the presence of one chlorine atom in the molecule. The HSQC and HMBC experiments revealed 14 carbon signals, which corresponded to five non-protonated carbon atoms,  -9 supported the presence of a proline moiety, while further interpretation of the HMBC data unambiguously connected the spin systems ( Figure 3) and verified the planar structure of 15. The NOE correlation of H-3 and H-6 determined their cis orientation and assigned the relative configuration of 15 that was identified as cis-cyclo(Pro-3-chloro-Tyr). Compound 15, described here for the first time as a natural product, has been previously reported as a synthetic derivative [43].
Compound 16, which also displayed two sodium adduct ion peaks at m/z 317.0657 and 319.0627 with a ratio of 3:1 (HRESIMS), was isolated as white solid. The spectroscopic characteristics of 16 (Tables 1 and 2 and Figures S7-S12) were rather similar to those of 15. Specifically, the NMR spectra of 16 revealed the same structural characteristics of a DKP moiety, including a proline amino acid and a 1,2,4-trisubstituted aromatic ring. The most prominent difference was that H-3 (4.13 ppm) and H-6 (3.22 ppm) resonated in higher fields, which, in combination with the absence of an NOE correlation between them, indicated that compound 16 was the trans isomer of 15. The COSY cross-peaks and the HMBC correlations observed for 16 ( Figure 3), in accordance to those observed for compound 15, were in agreement with the proposed structure of trans-cyclo(Pro-3-chloro-Tyr).
Compound 31, was obtained in trace amounts as a 1:1 mixture with compound 30. The gas chromatography -electron ionization mass spectrometry (GC-EIMS) chromatogram included two peaks, the first displaying a molecular ion peak [M] + at m/z 276 and a fragmentation pattern identical to that of cis-cyclo(Tyr-Ile) (30), whereas the second displayed molecular ion peaks [M] + at m/z 310 and 312 with an isotopic ratio of 3:1, suggesting that 31 was a monochlorinated compound. Comparison of the 1 H NMR data of the mixture with that of cis-cyclo(Tyr-Ile) (30) in pure form revealed the structural similarity of metabolites 31 and 30, with the main difference observed in the aromatic ring (Tables 1  and 2 and Figures S13-S17). Indeed, in the aromatic region of the 1 H NMR spectrum, the signals at δ Since several inconsistencies are observed for the published NMR data of frequently isolated DKPs, in conjunction to the fact that NMR data are incompletely reported for a number of them, the 1 H and 13 C NMR data for the known compounds 1-14, 17-30, and 32 are presented in Tables 1 and 2, complementing and revising the relevant literature data. Through careful analysis of the 13 C NMR chemical shifts of the proline-containing cis/trans pairs 4/5, 6/7, 8/9, 11/12, and 15/16, it can be observed that the chemical shifts of C-3 and C-10 are consistently deshielded by 3 and 3.5-4.5 ppm, respectively, in the trans DKP isomers.   [40], 13 [41], 17 [44], 21 [37], 23 [17], 27 [17], 29 [54]. 3 Determined through HMBC correlations. 4 Recorded at 100 MHz. 5  The COSY correlations of H-6/H2-7, H2-7/H2-8, and H2-8/H2-9 supported the presence of a proline moiety, while further interpretation of the HMBC data unambiguously connected the spin systems ( Figure 3) and verified the planar structure of 15. The NOE correlation of H-3 and H-6 determined their cis orientation and assigned the relative configuration of 15 that was identified as cis-cyclo(Pro-3-chloro-Tyr). Compound 15, described here for the first time as a natural product, has been previously reported as a synthetic derivative [43]. Compound 16, which also displayed two sodium adduct ion peaks at m/z 317.0657 and 319.0627 with a ratio of 3:1 (HRESIMS), was isolated as white solid. The spectroscopic characteristics of 16 (Tables 1 and 2 and Figures S7-S12) were rather similar to those of 15. Specifically, the NMR spectra of 16 revealed the same structural characteristics of a DKP moiety, including a proline amino acid and a 1,2,4-trisubstituted aromatic ring. The most prominent difference was that H-3 (4.13 ppm) and H-6 (3.22 ppm) resonated in higher fields, which, in combination with the absence of an NOE correlation between them, indicated that compound 16 was the trans isomer of 15. The COSY cross-peaks and the HMBC correlations observed for 16 (Figure 3), in accordance to those observed for compound 15, were in agreement with the proposed structure of trans-cyclo(Pro-3-chloro-Tyr).
Compound 31, was obtained in trace amounts as a 1:1 mixture with compound 30. The gas chromatography -electron ionization mass spectrometry (GC-EIMS) chromatogram included two peaks, the first displaying a molecular ion peak [M] + at m/z 276 and a fragmentation pattern identical to that of cis-cyclo(Tyr-Ile) (30), whereas the second displayed molecular ion peaks [M] + at m/z 310 and 312 with an isotopic ratio of 3:1, suggesting that 31 was a monochlorinated compound. Comparison of the 1 H NMR data of the mixture with that of cis-cyclo(Tyr-Ile) (30) in pure form revealed the structural similarity of metabolites 31 and 30, with the main difference observed in the aromatic ring (Tables 1 Table 2 and Figures S13-S17). Indeed, in the aromatic region of the 1 H NMR spectrum, the signals at  Compounds 15 and 16 were evaluated for their antifungal activity against Candida albicans and Aspergillus niger. However, neither of the two metabolites exerted any significant effect on the growth of the two fungal strains.

General Experimental Procedures
Optical rotations were measured on a Krüss model P3000 polarimeter (A. KRÜSS Optronic GmbH, Hamburg, Germany) with a 0.5 dm cell. UV spectra were obtained on a Perkin Elmer Lambda 40 spectrophotometer (PerkinElmer Ltd., Buckinghamshire, UK). IR spectra were obtained on a Bruker Alpha II spectrometer (Bruker Optik GmbH, Ettlingen, Germany). 1D and 2D NMR spectra were recorded on Bruker DRX 400, Avance NEO 700 and Avance NEO 950 (Bruker BioSpin GmbH, Rheinstetten, Germany) and Varian 600 (Varian, Inc., Palo Alto, CA, USA), spectrometers, using standard Bruker or Varian pulse sequences at room temperature. Chemical shifts are given on a δ (ppm) scale using TMS as internal standard. High-resolution electrospray ionization (ESI) mass spectra were measured on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Low-resolution electron ionization (EI) mass spectra were measured on a Hewlett-Packard 5973 mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) or on a Thermo Electron Corporation DSQ mass spectrometer (Thermo Electron Corporation

Biological Material
The bacterial strains were isolated from marine sediments collected from the East Mediterranean Sea and were identified based on comparison of their 16S ribosomal RNA (rRNA) sequences with data from the Genbank database of the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST). Specifically, strain BI0327, identified as Bacillus endophyticus

Fermentation, Extraction, and Isolation
The bacterial strain BI0327 was inoculated from a glycerol stock into a 1 L flask containing 500 mL of freshly prepared seawater-based (A1BFe+C) medium (10 g starch, 4 g yeast extract, 2 g peptone, 1 g CaCO 3 , 0.1 g KBr, and 0.04 g Fe 2 (SO4) 3 5H 2 O per liter of filtered seawater) [56]. After 7 days of incubation at 28 • C, while shaking at 120 rpm in an orbit shaker, the starter cultures were inoculated into 3 L flasks containing 1.5 L of the same seawater-based medium (4% v/v inoculum), to a total of 12 L of liquid medium, which were incubated at 28 • C for 14 days, while shaking at 120 rpm in an orbit shaker. Four days before the end of the fermentation period, Amberlite XAD-7HP resin (Sigma-Aldrich, St. Louis, MO, USA) (20 g/L) was added to each flask to adsorb extracellular metabolites. The broth was centrifuged and the pellet (resin and cell mass), was extracted twice for 24 h with Me 2 CO (8 L in total). Filtration of the extract and removal of the solvent under vacuum at 38 • C afforded a solid residue, which was partitioned between n-butanol and H 2 O. Evaporation of the solvent of the n-butanol soluble fraction in vacuo afforded a dark brown oily residue (2.2 g) that was subjected to vacuum column chromatography on silica gel, using cyclohexane, with increasing amounts of EtOAc, followed by EtOAc, with increasing amounts of MeOH as the mobile phase, to afford 8 fractions (327A-327H). Fraction 327G (50% MeOH in EtOAc, 1.1 g) was further fractionated by gravity column chromatography on silica gel, using EtOAc with increasing amounts of MeOH as the mobile phase, to yield 26 fractions (327G1-327G26). Fractions 327G10 to 327G15 (2% to 10% MeOH in EtOAc, 110.0 mg) were combined and purified by reversed-phase HPLC, using MeOH/H 2 O (70:30 and subsequently 50:50) and MeCN/H 2 O (30:70) as eluent, to afford 3 (3.9 mg), 4 (2.4 mg), 5 (1.7 mg), 6 (8.3 mg), 8 (6.6 mg), 11 (7.3 mg), 13 (6.1 mg), and 17 (1.2 mg). Fractions 327G16 (10% MeOH in EtOAc, 29.7 mg) and 327G17 (10% MeOH in EtOAc, 26.3 mg) were separately purified by reversed-phase HPLC, using MeOH/H 2 O (50:50) as eluent, and subsequently normal-phase HPLC, using cyclohexane/Me 2 CO (20:80) as eluent, to yield 7 (2.7 mg) and 13 (6.1 mg).
The bacterial strain BI0383 was inoculated from a glycerol stock into a 100 mL flask containing 50 mL of freshly prepared seawater-based (A1BFe+C) medium. After 4 days of incubation at 24 • C while shaking at 125 rpm in an orbit shaker, the starter culture was streaked onto 18 freshly prepared agar plates containing the same seawater-based medium. After 7 days, when sufficient growth of the bacterial strain was observed, mycelia were picked from the agar plates and were inoculated into 2 L flasks containing 1 L of the same seawater-based medium, to a total of 6 L of liquid medium, that were incubated at 27 • C for 7 days while shaking at 125 rpm in an orbit shaker. At the end of the fermentation period, Amberlite XAD-7HP resin (20 g/L) was added to each flask to adsorb extracellular metabolites. The culture and resin were shaken overnight at low speed. The broth was centrifuged and the pellet (resin and cell mass) was extracted twice for 24 h with Me 2 CO (4 L in total). Filtration of the extract and removal of the solvent under vacuum at 38 • C afforded a solid residue, which was partitioned between n-butanol and H 2 O. Evaporation of the solvent of the n-butanol soluble fraction in vacuo afforded a brown oily residue (2.22 g) that was subjected to vacuum column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc, followed by EtOAc with increasing amounts of MeOH as the mobile phase, to yield 12 fractions (383A-383L). Fraction 383K (10% MeOH in EtOAc, 54.0 mg) was submitted to normal-phase HPLC, using cyclohexane/Me 2 CO (55:45) as eluent, to afford 4 (6.0 mg), 3 (0.4 mg), and 8 (0.9 mg). The soluble in 80% MeOH in H 2 O part (111.0 mg) of fraction 383L (25% MeOH in EtOAc) was purified by reversed-phase HPLC, using MeCN/H 2 O (10:90) as eluent, to yield 1 (10.0 mg). The soluble in 50% MeOH in H 2 O part (77.0 mg) of fraction 383L was purified by reversed-phase HPLC, using MeCN/H 2 O (40:60) as eluent, to yield 6 (1.7 mg) and 13 (1.1 mg).
The bacterial strain BI0618 was streaked from a glycerol stock onto 25 freshly prepared agar plates containing a seawater-based (A1BFe+C) medium. After 3 days, when sufficient growth of the bacterial strain was observed, mycelia were picked from the agar plates and were inoculated into 1 L flasks containing 400 mL of the same seawater-based medium, to a total of 10 L of liquid medium, which were incubated at 24 • C for 8 days, while shaking at 130 rpm in an orbit shaker. At the end of the fermentation period, Amberlite XAD-7HP resin (20 g/L) was added to each flask to adsorb extracellular metabolites. The culture and resin were shaken overnight at low speed. The broth was centrifuged and the pellet (resin and cell mass) was extracted twice for 24 h with Me 2 CO (8 L in total). Filtration of the extract and removal of the solvent under vacuum at 38 • C afforded a solid residue, which was partitioned between EtOAc and H 2 O. Evaporation of the solvent of the EtOAc soluble fraction in vacuo afforded a dark orange oily residue (498 mg) that was subjected to vacuum column chromatography on silica gel, using cyclohexane, with increasing amounts of EtOAc, followed by EtOAc with increasing amounts of MeOH as the mobile phase, to yield 12 fractions (618A-618L). Fraction 618F (100% EtOAc, 8.4 mg) was identified as compound 4. Fraction 618G (5% MeOH in EtOAc, 40.1 mg) was subjected to normal-phase HPLC, using cyclohexane/Me 2 CO (55:45) as eluent, to afford compounds 4 (6.1 mg) and 8 (5.3 mg). Fraction 618H (20% MeOH in EtOAc, 140.1 mg) was subjected to vacuum column chromatography on silica gel, using cyclohexane with increasing amounts of Me 2 CO, followed by Me 2 CO with increasing amounts of MeOH as the mobile phase, to afford 10 fractions (618H1-618H10). The bacterial strain BI0918 was inoculated from a glycerol stock into a 100 mL flask containing 50 mL of freshly prepared seawater-based (A1BFe+C) medium. After 4 days of incubation at 24 • C, while shaking at 120 rpm in an orbit shaker, the starter culture was streaked onto 25 freshly prepared agar plates containing the same seawater-based medium. After 7 days when sufficient growth of the bacterial strain was observed, mycelia were picked from the agar plates and were inoculated into 1 L flasks containing 400 mL of the same seawater-based medium, to a total of 20 L of liquid medium, which were incubated at 24 • C for 8 days, while shaking at 120 rpm in an orbit shaker. At the end of the fermentation period, Amberlite XAD-7HP resin (20 g/L) was added to each flask to adsorb extracellular metabolites. The culture and resin were shaken overnight at low speed. The broth was centrifuged and the pellet (resin and cell mass) was extracted twice for 24 h with Me 2 CO (12 L in total). Filtration of the extract and removal of the solvent under vacuum at 38 • C afforded a solid residue, which was partitioned between EtOAc and H 2 O. Evaporation of the solvent of the EtOAc soluble fraction in vacuo afforded a dark red oily residue (2.0 g) that was subjected to vacuum column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc, followed by EtOAc with increasing amounts of MeOH as the mobile phase, to yield 14 fractions (918A-918N). The bacterial strain BI0980 was inoculated from a glycerol stock into two 100 mL flasks containing 50 mL of freshly prepared seawater-based (A1BFe+C) medium. After 5 days of incubation at 24 • C while shaking at 120 rpm in an orbit shaker, the starter cultures were inoculated into two 1 L flasks containing 500 mL of the same seawater-based medium (10% v/v inoculum) that were incubated at 24 • C for 4 days, while shaking at 120 rpm in an orbit shaker. Subsequently, they were inoculated into 1 L flasks containing 500 mL of the same seawater-based medium (10% v/v inoculum), to a total of 10 L of liquid medium, that were incubated at 24 • C for 9 days while shaking at 120 rpm in an orbit shaker. At the end of the fermentation period, Amberlite XAD-7HP resin (20 g/L) was added to each flask to adsorb extracellular metabolites. The culture and resin were shaken overnight at low speed. The broth was centrifuged and the pellet (resin and cell mass) was extracted twice for 24 h with Me 2 CO (6 L in total). Filtration of the extract and removal of the solvent under vacuum at 38 • C afforded a solid residue, which was partitioned between EtOAc and H 2 O. Evaporation of the solvent of the EtOAc soluble fraction in vacuo afforded a dark red oily residue (533.9 mg) that was subjected to vacuum column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc, followed by EtOAc with increasing amounts of MeOH as the mobile phase, to yield 14 fractions (980A-980N). Fraction 980J (5% MeOH in EtOAc, 26.8 mg) was purified by normal-phase HPLC, using cyclohexane/Me 2 CO (50:50) as eluent, to yield 4 (11.2 mg) and 8 (3.3 mg). Fraction 980K (10% MeOH in EtOAc, 51.8 mg) was repeatedly purified by normal-phase HPLC, using cyclohexane/acetone (30: cis-Cyclo(Pro-3-chloro-Tyr) (

Conclusions
The chemical investigation of the organic extracts of the fermentation broths of five marine-derived strains isolated from sediments collected from the East Mediterranean Sea resulted in the isolation and structure elucidation of three new 2,5-DKPs, namely cis-cyclo(Pro-3-chloro-Tyr) (15), trans-cyclo(Pro-3-chloro-Tyr) (16), and cis-cyclo(3-chloro-Tyr-Ile) (31). It is not unusual for marine macro-and microorganisms to incorporate halogens, mainly chlorine and bromine atoms, in their secondary metabolism, in order to increase the bioactivity of the compounds they biosynthesize [57,58]. Indeed, the brominated analogues of 15 and 16 have already been isolated from the actinobacterium Nocardia ignorata [59]. Additionally, the relevant literature is supplemented with complete NMR assignments and revisions for 29 previously reported 2,5-DKPs.