89 On the Isolation of 2-Hydroxydocosanoic and 2-Hydroxytricosanoic Acids From the Marine Sponge Arnphimedon compressa Nestor M. Carballeira* and Mario R. Lopez Department of Chemistry, University of Puerto Rico, Rio Piedras, Puerto Rico 00931 The first a-hydroxy fatty acids from a marine sponge, namely 2-hydroxydocosanoic and 2-hydroxytricosanoic, were identified in the Caribbean sponge Amphimedon compressa. These acids were found to occur in phospha- tidylethanolamine and phosphatidylserine and consti- tuted 52% of the total fatty acid mixture of this sponge. The long chain fatty acids 5,9,23-nonacosatrienoic (29:3) and 5,9,23-tricontatrienoic (30:3), as well as a new tetra- tricontatetraenoic (34:4) acid, were also found in A. compressa. Lipids 24, 89-91 (1989). Sponges have attracted in recent years the attention of marine natural products chemists because they have proved to be rich sources of bioactive and novel secon- dary metabolites. Unusual and unprecedented phospho- lipid fatty acids with no terrestrial counterpart have been isolated and characterized from dozens of marine sponges (1). However, one important group of phospholipid fatty acids, the 2-hydroxy acids, has not been recognized to exist in the phospholipids of marine sponges. One piece of work by Ayanoglu et al. (2), in which the phospholipid fatty acids of the marine sponge Higginsia tethyoides were analyzed, describes the isolation of a series of closely related 2-methoxy fatty acids, the only example to date of 2-oxa-substituted fatty acids in marine sponges. Unique to the latter work was the observation that these fatty acids were shown to be present in conventional phospholipids like phosphatidylethanolamine and phos- phatidylserine (2), when the normal expectation is for these acids to be present in sphingolipids. Four kinds of fatty acid occur in sphingolipids, the saturated long-chain fatty acids like palmitic (16:0), very long chain saturated fatty acids like behenic (22:0), monoenoic fatty acids such as oleic (18:1) and a-hydroxy very long chain fatty acids such as cerebronic (h 24:0). Important in this context is to mention that polyunsaturated fatty acids are con- spicuously absent from these lipids. In this paper we wish to report, for the first time, the isolation of the important fatty acids 2-hydroxydoco- sanoic {Scheme 1) and 2-hydroxytricosanoic {Scheme 2) from the phospholipids of the marine sponge Amphime- don compressa. This is the first time, to our knowledge, that these 2-hydroxy fatty acids have been isolated from conventional sponge phospholipids. Such hydroxylated acids usually are encountered in brain cerebrosides, sphin- gomyelins and lecithins (3) and in smaller concentrations in the kidney (4). *To whom correspondence should be addressed. Abbreviations: ~3 P, triphenylphosphine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; ECL, equivalent chain length; FT-NMR, Fourier transform nuclear mag- netic resonance; GC-MS, gas chromatography-mass spectrometry; PLC, preparative layer chromatography; 31p-NMR, phosphorus nuclear magnetic resonance; TLC, thin layer chromatography. O O U II (c.,),r_c. Ho/C~cH / I I OH OH SCHEME 1 SCHEME 2 EXPERIMENTAL PROCEDURES Amphimedon compressa was collected May 3, 1988, near La Parguera, Puerto Rico. The sponge was washed in sea water, carefully cleaned of all nonsponge debris and cut into small pieces. Immediate extraction with chloroform/ methanol (1:1, v/v) yielded the total lipids. The neutral lipids, glycolipids and phospholipids were separated by column chromatography using the procedure of Privett et al. (5). The phospholipid classes were investigated by thin layer chromatography (TLC) using silica gel and chloroform-methanol-water (75:25:4, v/v/v) as solvent. The 31p-NMR of the phospholipids was performed at 22~ on a GN 300 FT-NMR spectrometer at 121.6 MHz. For the acquisition, 16K data points were used and ca. 1000 accumulations were obtained before Fourier transforma- tion of the free induction decay. In a typical run, phos- pholipids {20-30 mg) were dissolved in 3 ml of deuterated chloroform-methanol (2:1, v/v) containing, as internal reference, triphenylphosphine (+3P). The fatty acyl com- ponents of the phospholipids were obtained as their methyl esters by reaction of the phospholipids with methanolic hydrogen chloride as described before (6). The resulting methyl esters were analyzed by gas chromatog- raphy-mass spectrometry (GC-MS) using a Hewlett- Packard 5995 A gas chromatograph-mass spectrometer equipped with a 30-m • 0.32-mm fused silica column coated with SE-54. For the location of double bonds, N- acylpyrrolidide derivatives were prepared by direct treat- ment of the methyl esters with pyrrolidine/acetic acid (10:1, v/v) in a capped vial (1 hr at 100~ followed by ethereal extraction from the acidified solution and purification by preparative layer chromatography (PLC). Hydrogenations were carried out in 10 ml of absolute methanol and catalytic amounts of platinum oxide (PtO2). Mass spectral data of key fatty acids for this discussion are presented below. 2-Hydroxydocosanoic acid methyl ester. MS m/z (rel in- tensity}: 370 (M +, 88), 348 (6), 338 (M + - MeOH, 5), 312 {13}, 311 (M + - COOMe, 56), 309 (9), 292 (9), 293 {2), 159 {6), 145 (10), 127 {15}, 125 {14}, 111 {27}, 103 (14}, 97 (48), 90 {38}, 83 {50}, 71 {38}, 69 (52}, 57 (100), 55 (81). 2-Hydroxytricosanoic acid methyl ester. MS rn/z {rel in- tensity}: 384 (M +, 80), 370 (4), 352 (M + - MeOH, 6), 338 (5), 326 {15}, 325 {M+ - COOMe, 62), 323 (17}, 306 {13}, 280 (3}, 159 (9), 145 (13), 127 (22}, 125 (18), 103 (14}, 90 (31), 83 {49}, 69 (49}, 59 {27}, 57 (100), 55 {84}. 5,9,23oNonacosatrienoic acid methyl ester. MS m/z (rel intensity}: 446 (M +, 7), 414 (2), 345 (5), 221 (4), 208 (4), LIPIDS, Vot, 24, No, 1 (1989)