Synthesis and antifungal properties of α-methoxy and α-hydroxyl substituted 4-thiatetradecanoic acids

4-Thiatetradecanoic acid exhibited weak antifungal activities against Candida albicans (ATCC 60193), Cryptococcus neoformans (ATCC 66031), and Aspergillus niger (ATCC 16404) (MIC = 4.8–12.7 mM). It has been demonstrated that α-methoxylation efficiently blocks β-oxidation and significantly improve the antifungal activities of fatty acids. We examined whether antifungal activity of 4-thiatetradecanoic acid can be improved by α-substitution. The unprecedented (±)-2-hydroxy-4-thiatetradecanoic acid was synthesized in four steps (20% overall yield), while the (±)-2-methoxy-4-thiatetradecanoic acid was synthesized in five steps (14% overall yield) starting from 1-decanethiol. The key step in the synthesis was the hydrolysis of a trimethylsilyloxynitrile. In general, the novel (±)-2-methoxy-4-thiatetradecanoic acid displayed significantly higher antifungal activities against C. albicans (ATCC 60193), C. neoformans (ATCC 66031), and A. niger (ATCC 16404) (MIC = 0.8–1.2 mM), when compared with 4-thiatetradecanoic acid. In the case of C. neoformans the (±)-2-hydroxy-4-thiatetradecanoic acid was more fungitoxic (MIC = 0.17 mM) than the α-methoxylated analog, but not as effective against A. niger (MIC = 5.5 mM). The enhanced fungitoxicity of the (±)-2-methoxy-4-thiatetradecanoic acid, as compared to decylthiopropionic acid, might be the result of a longer half-life in the cells due to a blocked β-oxidation pathway which results in more time to exert its toxic effects. Thus, these novel fatty acids may have applications as probes to study fatty acid metabolic routes in human cells

2,6‐hexadecadiynoic acid and 2,6‐nonadecadiynoic acid: Novel synthesized acetylenic fatty acids as potent antifungal agents

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 μM), and against Cryptococcus neoformans ATCC 66031 (MIC\u3c5.7 μM). 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 \u3c5.8 μM. 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 μM) against the fluconazole‐resistant C, albicans ATCC 14053 strain, but it showed a MIC value of only 100 μM against C. albicans ATCC 60193. 2,6‐Hexadecadiynoic acid and 2‐hexadecynoic acid also displayed a MIC of 140–145 μM 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

Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation.The work performed by the F.D.L.C. group was supported by grants BFU2011-26608 from the Spanish Ministry of Economy and Competitiveness and 612146/FP7-ICT-2013-10 and 282004/FP7-HEALTH-2011-2.3.1-2 from the European Seventh Framework Programme. The work performed by M.G. was supported by a Ph.D. fellowship from the University of Cantabria. The work performed by D.J.S.-R. was supported by the National Center for Research Resources and the National Institute of General Medical Sciences of the National Institutes of Health through grant no. 5P20GM103475-13 and the Interamerican University of Puerto Rico. The work performed by J.C.-G. was supported by an EMBO postdoctoral fellowship, ASTF 402-2010. The work performed by Biomar Microbial Technologies was supported by grant 282004/FP7-HEALTH-2011-2.3.1-2 from the European Seventh Framework Programme.USD 2,190 APC fee funded by the EC FP7 Post-Grant Open Access PilotPeer reviewe

Conjugation inhibitors compete with palmitic acid for binding to the conjugative traffic ATPaseTrwD, providing a mechanism to inhibit bacterial conjugation

Bacterial conjugation is a key mechanism by which bacteria acquire antibiotic resistance. Therefore, conjugation inhibitors (COINs) are promising compounds in the fight against the spread of antibiotic resistance genes among bacteria. Unsaturated fatty acids (uFAs) and alkynoic fatty acid derivatives, such as 2-hexadecanoic acid (2-HDA), have been reported previously as being effective COINs. The traffic ATPase TrwD, a VirB11 homolog in plasmid R388, is the molecular target of these compounds, which likely affect binding of TrwD to bacterial membranes. In this work, we demonstrate that COINs are abundantly incorporated into Escherichia coli membranes, replacing palmitic acid as the major component of the membrane. We also show that TrwD binds palmitic acid, thus facilitating its interaction with the membrane. Our findings also suggest that COINs bind TrwD at a site that is otherwise occupied by palmitic acid. Accordingly, molecular docking predictions with palmitic acid indicated that it shares the same binding site as uFAs and 2-HDA, although it differs in the contacts involved in this interaction. We also identified 2-bromopalmitic acid, a palmitate analog that inhibits many membrane-associated enzymes, as a compound that effectively reduces TrwD ATPase activity and bacterial conjugation. Moreover, we demonstrate that 2-bromopalmitic and palmitic acids both compete for the same binding site in TrwD. Altogether, these detailed findings open up a new avenue in the search for effective synthetic inhibitors of bacterial conjugation, which may be pivotal for combating multidrug-resistant bacteria.This work was supported by Spanish Ministerio de Economia y Competitividad (MINECO) Grants BFU2016-78521-R (to E. C. and I. A.) and BFU2014-55534 (to F. d. l. C.) and by Grant P20GM103475-16 from the National Center for Research Resources and NIGMS, National Institutes of Health (to D. S. R.). The authors declare that they have no conflicts of interest withthe contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health