Pseudoalteromone B: A Novel 15C Compound from a Marine Bacterium Pseudoalteromonas sp. CGH2XX

A novel 15C compound, pseudoalteromone B (1), possessing a novel carbon skeleton, was obtained from a marine bacterium Pseudoalteromonas sp. CGH2XX. This bacterium was originally isolated from a cultured-type octocoral Lobophytum crassum, that was growing in cultivating tanks equipped with a flow-through sea water system. The structure of 1 was established by spectroscopic methods. Pseudoalteromone B (1) displayed a modestly inhibitory effect on the release of elastase by human neutrophils.

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Introduction
Marine bacteria belonging to the genus Pseudoalteromonas sp. (family Pseudoalteromonadaceae) have proven to be not only an important source of various antibiotics, but have also played an interesting role in marine ecology [1][2][3][4]. In the continuing research aimed at the discovery of new natural substances from marine microorganisms, an organic extract of the bacterium identified as Pseudoalteromonas sp. CGH2XX, which was originally isolated from a cultured-type octocoral Lobophytum crassum (family Alcyonacea), exhibited significant cytotoxicity toward the HL-60 (human acute promyelocytic leukemia) and CCRF-CEM (human T cell acute lymphoblastic leukemia) tumor cells (IC 50 = 0.9, 1.2 µg/mL) and displayed a significant inhibitory effect (inhibition rate 45.1%) on the release of elastase by human neutrophils at a concentration of 10 µg/mL. We isolated a novel 15C compound, pseudoalteromone B (1) (Figure 1), from this microorganism. The structure of 1 was established by spectroscopic methods and this compound displayed a modestly inhibitory effect on the release of elastase by human neutrophils.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1020 polarimeter. IR spectra were recorded on a Jasco FT/IR-4100 infrared spectrophotometer. The NMR spectra were recorded on a Varian Mercury Plus 400 FT-NMR at 400 MHz for 1 H and 100 MHz for 13 C, in CDCl 3 , respectively. Proton chemical shifts were referenced to the residual CHCl 3 signal (δ H 7.26 ppm). 13 C NMR spectra were referenced to the center peak of CDCl 3 at δ C 77.1 ppm. ESIMS and HRESIMS data were recorded on a Bruker APEX II mass spectrometer. Silica gel (Merck, 230-400 mesh) and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography. TLC was carried out on precoated Kieselgel 60 F 254 (0.25 mm, Merck); spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating.

Marine Bacteria Isolation, Culture Conditions and Extract Preparation
A marine bacterium number CGH2XX was isolated from soft coral Lobophytum crassum that was growing in cultivating tanks equipped with a flow-through sea water system [4]. The bacterium strain CGH2XX was 98.3% identical with Pseudoalteromonas sp. H02P24-23 (Genebank accession no. HQ161380) on the basis of 16S rDNA gene sequence. The marine bacterium was cultured in 2.5 L flasks containing 1 L M1 broth (not containing agar) with 80% seawater. Flasks were incubated at 25 °C on a rotatory shaker at 120 rpm. After five days of incubation, extraction of the culture broth (10.0 L) with ethyl acetate (EtOAc, 2 × 10.0 L) yielded 1.71 g of crude extract. The extracts obtained were stored at −20 °C.

Separation
Crude extract was separated on Sephadex LH-20 and eluted using a mixture of dichloromethane and methanol (1:1) to yield 17 fractions. Fraction 6 was selected for further study and purified by silica gel, using a mixture of n-hexane and EtOAc (2:1) as a mobile phase to afford compound 1 (4.2 mg).

Conclusions
In a previous study [4], an ubiquinone derivative, pseudoalteromone A, was isolated from Pseudoalteromonas sp. CGH2XX, and this compound was found to be cytotoxic toward MOLT-4 (human acute lymphoblastic leukemia) and T-47D (human breast ductal carcinoma) cells (IC 50 = 3.8, 4.0 µg/mL) and displayed moderately inhibitory effects on the generation of superoxide anion and the release of elastase (inhibition rates 38.0, 20.2%) by human neutrophils at a concentration of 10 µg/mL [12]. However, as described in the beginning of this communication, the organic extract of Pseudoalteromonas sp. CGH2XX showed significant cytotoxicity and anti-inflammatory activity. At this stage, the results showed that pseudoalteromone B (1) displayed a modestly anti-inflammatory activity and this compound was not cytotoxic toward HCT116, K-562, HL-60, CCRF-CEM, T-47D and MDA-MB-231 cells. We suggested that the other active components exist in the other fractions. The possible activity for pseudoalteromone B (1) will be studied if we can get enough material from Pseudoalteromonas sp. CGH2XX. Furthermore, to the best of our knowledge, compounds pseudoalteromones A and B, were the first two compounds from the marine bacterium belonging to the genus Pseudoalteromonas associated with octocorals.