Theonellamide G, a Potent Antifungal and Cytotoxic Bicyclic Glycopeptide from the Red Sea Marine Sponge Theonella swinhoei

In our search for bioactive metabolites from marine organisms, we have investigated the polar fraction of the organic extract of the Red Sea sponge Theonella swinhoei. Successive chromatographic separations and final HPLC purification of the potent antifungal fraction afforded a new bicyclic glycopeptide, theonellamide G (1). The structure of the peptide was determined using extensive 1D and 2D NMR and high-resolution mass spectral determinations. The absolute configuration of theonellamide G was determined by chemical degradation and 2D NMR spectroscopy. Theonellamide G showed potent antifungal activity towards wild and amphotericin B-resistant strains of Candida albicans with IC50 of 4.49 and 2.0 μM, respectively. Additionally, it displayed cytotoxic activity against the human colon adenocarcinoma cell line (HCT-16) with IC50 of 6.0 μM. These findings provide further insight into the chemical diversity and biological activities of this class of compounds.


NOESY HMBC
The attachment of the galactose moiety at postion-3 of His moiety was apparent from the NOESY correlation of the anomeric proton at δ H 5.03 to H-2″″ and secured by its HMBC cross peaks to C-2″″ and C-4″″. The absolute configuration of 1 was determined by acid hydrolysis followed by chiral GC-MS and Marfey's analyses. Chiral GC-MS of the acid hydrolysate and LC-MS of the hydrolysate product of 1 derivatized with N-α-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide (Marfey's reagent) indicated the presence of L-Asn, L-alloThr, L-Ser, 2S-i-Ser, L-Phe, L-BrPhe, 2S,3R-HOAsn, and D-galactose. However, the absolute stereochemistry of Apoa, Hisala, and Ahd residues could not be determined. This stereochemical assignment was confirmed by comparison of NMR coupling constant values and chemical shifts with literature [7,15,18]. The E,E geometry of the olefinic double bonds Δ 4δ and Δ 4δ of Apoa was assigned on the basis of the intense NOESY cross peaks between δ H 5.12 (1H, m, 4-δH) and 6.47 (1H, d, J = 16.2 Hz, 4-δH) and between δ H 6.56 (1H, d, J = 16.2 Hz, 4-εH) and 1.63 (3H, s, 4-εCH 3 ) and confirmed by the coupling constant values and the 13 C chemical shift of 4-εCH 3 (δ C 13.4). In conclusion, comparison of the spectral data of 1 with those of theonellamide A suggested the replacement of the β-MeBrPhe and α-amino-γ-hydroxyadipic acid in theonellamide A with BrPhe and Ahd in 1. Thus, the structure of 1 was unambiguously elucidated as depicted and the trivial name theonellamide G was given to it.
Theonellamide G (1) showed potent antifungal activity towards wild and amphotericin B-resistant strains of Candida albicans with IC 50 of 4.49 and 2.0 μM, respectively, compared to 1.48 μM for the positive antifungal control amphotericin-B against the wild type (Table 2). Additionally, compound 1 displayed cytotoxic activity against the human colon adenocarcinoma cell line (HCT-16) with IC 50 of 6.0 μM, compared to 2.0 μM for etoposide (positive anticancer control) ( Table 2).

Animal Materials
The marine sponge was collected by scuba diving at a depth of 4-5 m of Hurghada in the Red Sea coast. The sponge is cylindrical in shape and dark red-brown in color. The cut-off fragment measures 7.5 cm high and 4.5 cm in diameter. It has a central canal of 1.5 cm diameter leading to a narrow vent with sphincter-like membrane at the top. The in-situ photo shows the vent to be similar in diameter as the central canal. The surface is slightly bumpy, generally smooth, but furrowed lengthwise. The ectosomal skeleton consists of a dense mass of curved acanthomicrorhabds of 15-24 × 2-3 μm, overlying a lose reticulation of reduced phyllotriaenes with cladome spanning 120-180 μm and thin undivided cladi 55-120 × 4-7 μm in size. A subectosomal region measuring about 1 mm in thickness bridges an area devoid of desmas, the skeleton of which consists of bundles of strongylotes, measuring 25-70 μm in diameter, enclosing 4-20 strongylotes. The latter are slightly anisotylote with either end more or less swollen, 405-620 × 3-6 μm in size. The choanosomal skeleton consists of a loose reticulation of tetraclone desmas strengthened by bundles of strongylotes. Desmas cladomes measure 400-550 μm, rhabds smooth, 120-230 × 15-20 μm, and cladi smooth with simple zygoses, 150-250 × 12-16 μm. Compared with the type specimen there are some differences (lighter skeletal, smooth instead of tuberculated desmas, shorter strongylotes) which are judged to be infraspecific variation. The voucher fragment is registered in the collection of the Zoological Museum of Amsterdam under registration number POR 16637 and in the Red Sea Invertebrates Collection at Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt, under registration number DY-RS-59.

Extraction and Purifications of Compound 1
The frozen sponge materials (1.5 kg, wet weight) were extracted with a mixture of MeOH/CH 2 Cl 2 (1:1) (3 × 1000 mL) at room temperature. The combined extracts were concentrated under reduced pressure and suspended in MeOH/H 2 O (9:1) (1000 mL). The resulting mixture was extracted with n-hexane (3 × 400 mL) to give 7.2 g of n-hexane residue. The remaining methanolic layer was diluted with H 2 O to (3:2) MeOH/H 2 O and then extracted with CH 2 Cl 2 (3 × 400 mL) to give 2.4 g of CH 2 Cl 2 residue. The CH 2 Cl 2 residue was subjected to a Sephadex LH-20 column (Merck, Darmstadt, Germany) using MeOH as an eluent to afford nine fractions. Fraction 4 (730 mg) was subjected to ODS flash chromatography starting with 30% aqueous MeOH through pure MeOH to afford 10 subfractions. The potent antifungal subfraction eluted with 40% H 2 O in MeOH (subfraction 4) (86 mg) was subjected to final HPLC purification on a preparative C30 column (Develocil, C30-UG-5, 250 × 20 mm, Nomura Chemical, Setouchi-shi, Japan) using 25% n-propanol in water at a flow rate of 5.5 mL/min to afford compound 1 (11.5 mg).

Acid Hydrolysis and Absolute Configuration of Amino Acids Using LC-MS Analysis of the Marfey Derivatives of 1
Compound 1 (1.0 mg) was treated with 2 mL 6 N HCl (pa) and heated in sealed ampoule at 110 °C for 24 h under N 2 gas. The resulting solution was concentrated, with consecutive addition of H 2 O (5 mL) to ensure complete elimination of HCl. To 50 μL of acid hydrolysate (or authentic amino acid standard at comparable concentration), 100 μL FDNPL (1% N-(5-flouro-2,4-dinitrophenyl)-L-leucinamide in acetone) and 20 μL 1 M NaHCO 3 were added. The mixture was heated at 40 °C for 1 h over a hot plate with frequent mixing. After cooling, 10 μL of 2 M HCl was added and then concentrated to dryness before dissolving in 1000 μL MeOH. Standards of L and D amino acids were treated separately with FDNPL in the same manner. The FDNPL derivatives were analyzed using LC-MS by comparison of the retention time and molecular weight with those of standard amino acids FDNPL derivatives [22,23].

Chiral GC-MS Analysis of 1
About 0.2 mg of 1 was placed in sealed ampoule containing 6 N HCl (0.5 mL) and heated at 110 °C for 12 h. After evaporation of the solvent under a stream of N 2 gas, the residue was dissolved in 10% HC1/MeOH and heated at 100 °C for 30 min. The product was evaporated, dissolved in trifluoroacetic anhydride (50 μL) and CH 2 C1 2 (50 μL), reacted at 100 °C for 10 min, and evaporated in a stream of N 2 gas. The residue was dissolved in EtOAc (100 μL). Aliquot