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In the present study, the structural characteristics that impart antibacterial activity to C16 alkynoic fatty acids (aFA) were further investigated. The syntheses of hexadecynoic acids (HDA) containing triple bonds at C-3, C-6, C-8, C-9, C-10, and C-12 were carried out in four steps and with an overall yield of 34-78%. In addition, HDA analogs containing a sulfur atom at either C-4 or C-5 were also prepared in 69-77% overall yields, respectively. Results from this study revealed that the triple bond at C-2 is pivotal for the antibacterial activity displayed by 2-HDA, while the farther the position of the triple bond from the carbonyl group, the lower its bactericidal activity against gram-positive bacteria, including clinical isolates of methicillin-resistant Staphylococcus aureus (CIMRSA) strains. The potential of 2-HDA as an antibacterial agent was also assessed in five CIMRSA strains that were resistant to Ciprofloxacin (Cipro) demonstrating that 2-HDA was the most effective treatment in inhibiting their growth when compared with either Cipro alone or equimolar combinations of Cipro and 2-HDA. Moreover, it was proved that the inhibition of S. aureus DNA gyrase can be linked to the antibacterial activity displayed by 2-HDA. Finally, it was determined that the ability of HDA analogs to form micelles can be linked to their decreased activity against gram-positive bacteria, since critical micellar concentrations (CMC) between 50 and 300 μg/mL were obtained.

The naturally occurring (6Z)-(±)-2-methoxy-6-hexadecenoic acid (1) and (6Z)-(±)-2-methoxy-6-octadecenoic acid (2) were synthesized in 7–8 steps with 38 and 13% overall yields, respectively, by using an acetylide coupling approach, which made it possible to obtain a 100% cis-stereochemistry for the double bonds. In a similar fashion, the acetylenic analogs (±)-2-methoxy-6-hexadecynoic acid (3) and (±)-2-methoxy-6-octadecynoic acid (4) were also synthesized in 6–7 steps with 48 and 16% overall yields, respectively. The antibacterial activity of acids 1–4 was determined against clinical isolates of methicillin-resistant Staphylococcus aureus (ClMRSA) and Escherichia coli. Among the series of compounds, acid 4 was the most active bactericide towards CIMRSA displaying IC50s (half maximal inhibitory concentrations) between 17 and 37 μg/mL, in sharp contrast to the 6-octadecynoic acid, which was not bactericidal at all. On the other hand, acids 1 and 3 were the only acids that displayed antibacterial activity towards E. coli, but 1 stood out as the best candidate with an IC50 of 21 μg/mL. The critical micelle concentrations (CMCs) of acids 1–4 were also determined. The C18 acids 2 and 4 displayed a five-fold lower CMC (15–20 μg/mL) than the C16 analogs 1 and 3 (70–100 μg/mL), indicating that 4 exerts its antibacterial activity in a micellar state. None of the studied acids were inhibitory towards S. aureus DNA gyrase discounting this type of enzyme inhibition as a possible antibacterial mechanism. It was concluded that the combination of α-methoxylation and C-6 unsaturation increases the bactericidal activity of the C16 and C18 FA towards the studied bacterial strains. Acids 1 and 4 stand out as viable candidates to be used against E. coli and CIMRSA, respectively.

The phospholipid fatty acid composition of the Caribbean sponge Erylus goffrilleri is described for the first time. A total of 70 fatty acids with chain lengths between 13 and 29 carbons were identified in the sponge. Methyl-branched fatty acids predominated in E. goffrilleri suggesting the presence of a considerable number of bacterial symbionts. The novel fatty acids (5Z,9Z)-2-methoxy-5,9-hexadecadienoic acid, (5Z,9Z)-2-methoxy-5,9-octadecadienoic acid, (5Z,9Z)-2-methoxy-5,9-nonadecadienoic acid, and (5Z,9Z)-2-methoxy-5,9-eicosadienoic acid are described for the first time in the literature. In addition, the iso-methyl-branched fatty acids (9Z)-2-methoxy-15-methyl-9-hexadecenoic acid and (5Z,9Z)-2-methoxy-15-methyl-5,9-hexadecadienoic acid, also identified in E. goffrilleri, were identified for the first time in nature. Based on the identified metabolites it is proposed that the unprecedented biosynthetic sequence: i-17:1Delta9 --2-OMe-i-17:1Delta9 --2-OMe-i-17:2Delta5,9 might be responsible for the biosynthesis of the novel iso-alpha-methoxylated fatty acids in E. goffrilleri.

The cyclopropane fatty acids 17-methyl-trans-4,5-methyleneoctadecanoic acid, 18-methyl-trans-4,5-methylenenonadecanoic acid, and 17-methyl-trans-4,5-methylenenonadecanoic acid were characterized for the first time in nature in the phospholipids (mainly PE, PG and PS) of the hermit-crab sponge Pseudospongosorites suberitoides. Pyrrolidine derivatization was the key in identifying the position of the cyclopropyl and methyl groups in the acyl chains and (1)H NMR was used to determine the trans stereochemistry of the cyclopropane ring. The phospholipids from the sponge also contained an interesting series of iso-anteiso Delta(5,9) fatty acids with chain-lengths between 17 and 21 carbons, with the fatty acids (5Z,9Z)-18-methyl-5,9-nonadecadienoic acid and the (5Z,9Z)-17-methyl-5,9-nonadecadienoic acid being described for the first time in sponges. The anteiso alpha-methoxylated fatty acid 2-methoxy-12-methyltetradecanoic acid was also identified for the first time in nature in the phospholipids of this interesting marine sponge. The novel cyclopropyl fatty acids could have originated from the phospholipids of a cyanobacterium living in symbiosis with the sponge.

The hitherto unknown 2,6-hexadecadiynoic acid, 2,6-nonadecadiynoic acid, and 2,9-hexadecadiynoic acid were synthesized in two steps and in 11-18% overall yields starting from either 1,5-hexadiyne or 1,8-nonadiyne. Among all the compounds 2,6-hexadecadiynoic acid displayed the best overall antifungal activity against both the fluconazole-resistant Candida albicans strains ATCC 14053 and ATCC 60193, with a minimum inhibitory concentration (MIC of 11 microM), and against Cryptococcus neoformans ATCC 66031 (MIC < 5.7 microM). 2,9-Hexadecadiynoic acid did not display any significant cytotoxicity against the fluconazole-resistant C. albicans strains, but it showed fungitoxicity against C. neoformans ATCC 66031 with a MIC value of < 5.8 microM. Other FA, such as 2-hexadecynoic acid, 5-hexadecynoic acid, 9-hexadecynoic acid, and 6-nonadecynoic acid were also synthesized and their antifungal activities compared with those of the novel acetylenic FA. 2-Hexadecynoic acid, a known antifungal FA, exhibited the best antifungal activity (MIC = 9.4 microM) against the fluconazole-resistant C. albicans ATCC 14053 strain, but it showed a MIC value of only 100 microM against C. albicans ATCC 60193. 2,6-Hexadecadiynoic acid and 2-hexadecynoic acid also displayed a MIC of 140-145 microM toward Mycobacterium tuberculosis H37Rv in Middlebrook 7H12 medium. In conclusion, 2,6-hexadecadiynoic acid exhibited the best fungitoxicity profile compared with other analogues. This diynoic FA has the potential to be further evaluated for use in topical antifungal formulations.

The phospholipid fatty acid composition of the sponge Amphimedon complanata was reinvestigated, and the 2-methoxy-13-methyltetradecanoic acid, 2-methoxy-14-methylpentadecanoic acid, and 2-methoxy-13-methylpentadecanoic acid were identified for the first time in nature. Structure characterization was accomplished by means of gas chromatographic retention times and gas chromatography-mass spectrometry. These acids could have originated from bacteria in symbiosis with the sponge.

The lipid composition of a Bacillus sp., isolated from Lake Pomorie in Bulgaria, was unusual and consisted of 26 different fatty acids between C12 and C26, with anteiso C15−C17 saturated fatty acids predominating. The furan fatty acid, 10,13-epoxy-11-methyloctadeca-10,12-dienoic acid, was also identified, a new finding for this genus. The hydrocarbons consisted of 30 different monounsaturated hydrocarbons, between C25 and C30, with the iso-iso, iso-anteiso, anteiso-anteiso, iso-normal, and anteiso-normal methyl branching for odd-numbered chains, and the iso-iso, iso-anteiso, iso-normal, and anteiso-normal methyl branching for even-numbered chains. The double bond positions in these hydrocarbons were determined by dimethyl disulfide derivatization followed by GC-MS, and the double-bond cis configuration was confirmed by infrared spectroscopy. Some previously unknown hydrocarbons in bacteria, such as (Z)-3,21-dimethyl-9-tricosene, (Z)-3,21-dimethyl-10-tricosene, (Z)-2,24-dimethyl-11-pentacosene, and (Z)-2,25-dimethyl-13-hexacosene were identified. Sterols were detected and were based on the sitosterol nucleus.

The fatty acid composition of the tunicate Botryllus schlosseri and of two bacterial strains found within the tunicate, namely Vibrio parahaemolyticus and of an associated but previously unreported gram positive cocci were studied. The polyunsaturated fatty acids 6,9,12-octadecatrienoic acid, 5,8,11,14,17-eicosapentaenoic acid, and 4,7,10,13,16,19-docosahexaenoic acid were particularly abundant in B. schlosseri and were not detected in the two bacterial strains found in the tunicate. The iso/anteiso pair, 13-methyltetradecanoic acid and 12-methyltetradecanoic acid, were the principal fatty acids in the gram positive cocci, and the 9- and 11-hexadecenoic acids were particularly abundant in V. parahaemolyticus. The diunsaturated fatty acid 9,12-octadecadienoic acid was also shown to be present in V. parahaemolyticus. The fatty acid composition of a third bacterial strain, characterized as either a Pseudomonas or an Alteromonas species, and shown to be present only in the sea water from the Black Sea and not in B. schlosseri, is also reported. This is the first investigation on fatty acids from Black Sea bacteria.

The distribution of C14-C22 fatty acids in the phosphatidylethanolamines isolated from the sponges Agelas sp. and Spongia tampa was investigated. Selective changes with phospholipase A2 (from Agkistrodon halys blomhoffii) followed by thin-layer chromatographic separation of the resulting lysophosphatidylethanolamines and free fatty acids and subsequent methylation with HCl/MeOH and diazomethane, respectively, revealed that the 5,9-octadecadienoic acid and the 9,12-octadecadienoic acid present showed no preference for either position sn-1 or sn-2 in these phosphatidylethanolamines. The other saturated and unsaturated fatty acids with chains between 14 and 22 carbons long were also found to be equally distributed between positions sn-1 and sn-2 in the phosphatidylethanolamines in these sponges. The results contrast with what is known about the distribution in most mammalian phospholipids, such as the phosphatidylcholines from human erythrocytes, where octadecadienoic acid typically occupies the sn-2 position.

The very long-chain fatty acids, (5E,9Z)-6-bromo-5,9-tetracosadienoic, (5E,9Z)-6-bromo-23-methyl-5,9-tetracosadienoic, (5E,9Z)-6-bromo-5,9-pentacosadienoic and (5E,9Z)-6-bromo-24-methyl-5,9- pentacosadienoic acids, were identified in the phospholipids (mainly phosphatidylethanolamine) of the sponge Agelas sp. Structure elucidation was accomplished by means of mass spectrometry and chemical transformations, including deuteration with Wilkinson's catalyst. All of the sterols from the sponge had the delta 5,7 nucleus, with 24-methylcholesta-5,7,22-trien-3 beta-ol (ergosterol) and 24-ethyl-cholesta-5,7, 22-trien-3 beta-ol being the most abundant.

The phospholipid fatty acids from the sponge Spheciospongia cuspidifera were studied revealing the presence of the rare 10-octadecenoic acid (10-18:1) and a new 2-methoxyhexadecenoic acid. The phospholipid fatty acids from Thalysias juniperina were also studied revealing the presence of the hitherto unreported 18-hexacosenoic acid (18-26:1). These results tend to indicate that the biosynthetic pathway from 10-18:1 to 18-26:1 may be operative in nature. The phospholipid mixture from the sponges was also analyzed by 31P-NMR and shown to mainly consist of phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Phosphatidylcholine was not found in the sponges analyzed in this work.