Antibacterial Secondary Metabolites from the Cave Sponge Xestospongia sp

Chemical investigation of the cave sponge Xestospongia sp. resulted in the isolation of three new polyacetylenic long chain compounds along with two known metabolites. The structures of the new metabolites were established by NMR and MS analyses. The antibacterial activity of the new metabolites was also evaluated.

In the course of our search for bioactive metabolites from sponges, we have investigated the CH 2 Cl 2 /MeOH (1:1 v/v) extract of the cave sponge Xestospongia sp. collected from Pohnpei, Federated States of Micronesia, which exhibited antimicrobial activity against the gram-negative bacteria Pseudomonas aeruginosa and the gram-positive bacteria Mycobacterium intracellulare with IC 50 's of 2.07 and 1.03 μg/mL, respectively. In this article we report the isolation and structure OPEN ACCESS elucidation of three new metabolites (1-3) along with two known metabolites (4, 5) ( Figure 1) which belong to the class of polyacetylenic long chain compounds, as well as their antibacterial activities.

Bioassay-Guided Isolation
The crude DCM extract of the sponge which displayed antibacterial activity was subjected to reverse phase HPLC (Phenomenex, Luna C 18 (2)) using a gradient mixture (94:6 CH 3 CN:H 2 O to 100% CH 3 CN over 43 min) to afford two pure compounds (4 and 5). Rest of the peaks were further separated on a PhenylHexyl column (Phenomenex, Luna, 250 × 10 mm, 5 μm) using an isocratic elution system (80:20 CH 3 CN:H 2 O) to yield compounds (1-3).  Compound 2 was obtained as colorless oil, had a molecular formula of C 22 H 38 O 2 as indicated by HRESIMS at m/z 357.2761 [M + Na] + . This suggested four degrees of unsaturation which were satisfied by two acetylene groups. The structure of compound 2 was similar to that of 1 except for the absence of double bond, and the terminal isopropyl group being replaced by a terminal methyl group at δ H 0.88 (3H, t), δ C 14.3 (CH 3 ). Thus the structure of the new compound was established as 2.

Structural Elucidation of the New Compounds
Compound 3 was isolated as colorless solid, had a molecular formula of C 24 H 40 O 2 as deduced by HRESIMS at m/z 383.2921 [M + Na] + which indicated five degrees of unsaturation. In addition to the two triple bonds, one primary and one secondary hydroxy moieties three mutually coupled high field signals were observed in the 1 H NMR spectrum [δ H −0.35 (1H, m), 0.55 (1H, m), δ C 10.8 and δ H 0.63 (2H, m), δ C 15.8] which were diagnostic for 1,2-disubstituted cyclopropane ring. The large difference in chemical shifts of the methylene group of the cyclopropane ring indicated cis stereochemistry of the three-membered cyclopropane ring which was confirmed by comparing 1 H NMR values with reported data [16]. The position of the cyclopropane ring could not be determined as no HMBC correlations were seen from the cyclopropyl proton signals to the triple bond or to the long chain terminal methyl group which indicated that the cyclopropane moiety is present in the side chain not closer to any of the above mentioned moieties. Also due to very limited amounts of the isolated compound no chemical reactions could be carried out in order to determine the position of the cyclopropane ring. However this is the first report of isolation of cyclopropane containing long chain acetylenic alcohol.
Compounds 4 and 5 were identified as known compounds 18-hydroxyrenierin-2 [17] and strongylodiol A [15] by comparison of spectral data to the literature. Comparison of the specific rotation of compound 5 to that of the reported value indicated R configuration at C-6. By analogy the sign of specific rotation of all other compounds (1, 2 and 4) also indicate R-configuration at C-6, which could be also true for compound 3 as it belongs to the same series of compounds. The isolated compounds were evaluated for antibacterial activity against P. aeruginosa and M. intracellulare and the results are presented in Table 2.

General
Optical rotations were measured using a JASCO DIP-370 digital polarimeter. UV spectra were recorded on a Hewlett-Packard 8452A diode array spectrometer. IR spectra were recorded on an ATI Mattson Genesis series FTIR spectrometer.NMR spectra were measured on Bruker Advance DRX-400 spectrometer. 1 H and 13 C NMR spectra were measured and reported in ppm using CDCl 3 solvent peak (δ H 7.24 and δ C 77.23) as an internal standard. ESI-FTMS analyses were measured on a Bruker Magnex BioAPEX 30es ion cyclotron HR HPLC-FT spectrometer by direct injection into an electrospray interface. EIMS data was acquired on Waters VG 70-250S magnetic sector mass spectrometer. HPLC purifications were carried out on a Waters 2695 model system equipped with dual absorbance UV detector.

Sponge Material
The sponge (ID: PN10407137) was collected from a small cave at a depth of about 40 m. The samples exuded pigment and were a crème color. They were massive in shape with a "velvet-like" feel to their surface that was somewhat brittle upon collection. This new species is currently being identified by the Porifera Tree of Life (PorToL) project. Voucher specimen of the sample was deposited at the NOAA Ocean Biotechnology Center and Repository, Oxford, MS, USA.

Antibacterial Assay
Organisms were obtained from the American Type Culture Collection (Manassas, VA, USA) which includes Pseudomonas aeruginosa ATCC 27853, and Mycobacterium intracellulare ATCC 23068. Susceptibility testing was performed using a modified version of the CLSI (formerly NCCLS) methods [18,19]. M. intracellulare was tested using a modified method of Franzblau et al. [20]. Samples were serially-diluted in 20% DMSO/saline and transferred in duplicate to 96 well flat bottom microplates. Microbial inocula were prepared by correcting the OD 630 of microbe suspensions in incubation broth to afford final target inocula. Ciprofloxacin (ICN Biomedicals, Aurora, OH, USA) was included as the positive control. Organisms were read at 530 nm using the Biotek Powerwave XS plate reader (Bio-Tek Instruments, Winooski, VT, USA) (P. aeruginosa) or 544ex/590em, (M. intracellulare) using the Polarstar Galaxy Plate Reader (BMG Lab Technologies, Germany) prior to and after incubation. IC 50 's (concentrations that afford 50% inhibition relative to controls) are calculated using XLfit 4.2 software (IDBS, Alameda, CA, USA, 2005) using fit model 201.

Conclusions
In summary, three new (1-3) and two known (4, 5) metabolites were isolated from the sponge Xestospongia sp. and their structural elucidation was done using spectroscopic analysis. These metabolites exhibited moderate antibacterial activity against gram-negative bacteria P. aeruginosa and the gram-positive bacteria M. intracellulare when compared to the positive control.
Bharathi Avula, National Center for Natural Products Research for providing NMR and mass spectral data respectively, Marsha Wright for biological testing. Our thanks to Deborah Gochfeld for sponge sample collection.