Marine Sponge Dysidea herbacea revisited: Another Brominated Diphenyl Ether

A pentabrominated phenolic diphenyl ether (1) that has not previously been reported from marine sources has been isolated from Dysidea herbacea collected at Pelorus Island, Great Barrier Reef, Australia. The structure was determined by comparison of NMR data with those of known structurally-related metabolites. NMR spectral assignments for (1) are discussed in context with those of three previously reported isomeric pentabrominated phenolic diphenyl ethers.


Introduction
It is well documented that the marine sponge Dysidea herbacea occurs in two general chemotypes: one produces sesquiterpenes (usually furanosesquiterpenes) and polychlorinated amino acid derivatives, while the other produces only polybrominated diphenyl ethers [1]. The production of the chlorinated metabolites and the polybrominated diphenyl ethers has been reported to be due to the filamentous cyanobacterium Oscillatoria spongeliae [2][3][4]. Recently, 16S-rDNA studies on Oscillatoria strains isolated from Dysidea species that exhibited different colourmorph and growth characteristics indicated that each species of Dysidea hosted a distinct strain of Oscillatoria, which was interpreted to imply a high degree of host specificity and possible coevolution between the symbiotic bacterium and its host sponge [5]. Recent studies have also reported detection of polybrominated diphenyl ethers in higher trophic groups such as fish, turtles, birds and even marine mammals [6,7a-c], implying that these compounds are bioaccumulated in nature, and may persist in significant concentrations in such higher trophic organisms.
As a group, the polybrominated diphenyl ethers exhibit a wide range of activities in bioassays, ranging from antibacterial activity (against S. aureus and T. mentagrophytes), to cytotoxicity (Ehrlich ascite tumor cells) [8]. This cytotoxicity is exhibited by inhibition of a range of enzymes that are implicated in tumor development, such as inosine monophosphate dehydrogenase, guanosine monophosphate synthetase and 15-lipoxygenase [9]. We previously reported the isolation of the pentabrominated diphenyl ether (2) from samples of Dysidea herbacea collected from Cattle Bay, Orpheus Island [10]. We now report the characterisation of the last remaining isomer of this particular group of pentabrominated phenolic diphenyl ethers. It was obtained as a minor metabolite from a Dysidea herbacea sample, that also contained (2) as its major metabolite, collected from Pelorus Island.

Results and Discussion
The 1 H NMR spectrum of the crude dichloromethane extract from a Dysidea herbacea sample collected from -11m at Pelorus Island in December 2003 contained, in addition to the signals characteristic of the pentabrominated diphenyl ether (2) [10], a singlet at δ7.74 indicative of the presence of a minor metabolite. The brominated diphenyl ether fraction was isolated by vacuum liquid chromatography, and the major and minor metabolites were separated by reverse-phase HPLC. The minor metabolite (1) was found by high-resolution electrospray mass spectrometry (negative ion mode) to have the same molecular formula as (2), C 12 H 5 Br 5 O 2 . The 1 H and 13 C NMR spectra were consistent with a phenolic diphenyl ether that contained a tribrominated phenolic A-ring, and a dibrominated B-ring. This meant that the A ring substitution pattern was isomeric with that of (2), but two (3 and 4) of the other three positional isomers for the sole hydrogen atom on the A-ring had already been reported from Dysidea and Phyllospongia samples [9,11]. The 1 H NMR signal for the sole hydrogen atom on ring A for (1) resonated at δ7.74, but at δ7.42 for (3) [10,11], δ7.01 for (4) [7] and δ7.55 for (2) [10] ( Table 1).
During our isolation and structural elucidation of this metabolite, a report of the synthesis of 1hydroxy-3,4,6,2',4'-pentabromodiphenyl ether was published as a full paper, elaborating on results that had previously been presented at the Dioxin 2001 meeting [7a,7b,7c]. However, only 1 H NMR data was presented in those reports. The reported 1 H NMR data is in agreement with that observed for (1).

Conclusions
The pentabrominated phenolic diphenyl ether (1) that has not previously been reported from marine sources has been isolated from Dysidea herbacea collected at Pelorus Island, Great Barrier Reef, Australia. This means that all 4 positional isomers of diphenyl ethers that contain a 2,4-dibrominated B-ring and a 1-hydroxytribrominated A-ring with the ether linkage at the 2-position have now been reported from marine sponges.

Acknowledgements
We wish to thank Dr. John Hooper from the Queensland museum for taxonomy and Mr. Rick Willis from A.I.M.S. for mass spectrometry.

Experimental
General IR spectra were determined on a Nicolet Nexus 670 infrared spectrometer. Mass spectral data were determined on a Bruker BioAPEX 47e mass spectrometer operating in negative ion electrospray mode at the Australian Institute of Marine Science, Cape Ferguson. 1 H NMR spectra were measured in CDCl 3 at 300 MHz and 13 C NMR spectra at 75.5 MHz on a Varian Mercury NMR using residual solvent peaks for calibration. Merck t.l.c. grade silica gel 5-40µ (type 60) was used for column chromatography. HPLC purification was carried out on a Hewlett-Packard C18 column (10 x 250 mm), monitored with a GBC diode array detector. The metabolite ratio was determined by integration at 292 nm. All solvents used were freshly distilled.

Animal material
The sponge Dysidea herbacea was collected by hand using scuba (from -11m) near Pelorus Island (18° 34´ S; 146° 29´ E) in the central section of the Great Barrier Reef Marine Park, Australia. The sample was frozen immediately after collection and kept frozen until used. A taxonomic sample (registered sample No.G25097) is lodged with the Museum of Tropical Queensland, Flinders Street, Townsville, Qld. 4810.

Extraction and Purification.
The freeze-dried sponge (10.89 g) was successively extracted with dichloromethane (3 × 100 ml). The solvent was removed on a rotary evaporator to afford a crude extract (0.448g) which was rapidly chromatographed on silica gel under vacuum using a stepwise gradient from hexane to dichloromethane to ethyl acetate. A mixture of the diphenyl ethers (1 and 2) (22.8mg, 0.21%) was eluted in the dichloromethane/hexane 1:9 and 1:4 fractions as a crystalline solid. This material was separated by reverse phase HPLC by elution with acetonitrile / 1% aqueous ammonium acetate (9:1) at a flow rate of 1.5 ml/min. The acetonitrile was removed from fractions that contained the metabolites 1 (retention time 3.23 min ) and 2 (retention time 2.85 min ) and each was transferred to a separating funnel and extracted with dichloromethane. Removal of the dichloromethane solvent afforded the minor metabolite 1 and the major metabolite 2 in a ratio of 1:10 (based on integrated peak areas). Hydroxy-3,4,6,2