- Pitfalls in the sample preparation and analysis of N-acylethanolamines
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N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and 2-arachidonoylglycerol existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data. Copyright
- Skonberg, Christian,Artmann, Andreas,Cornett, Claus,Hansen, Steen Honore,Hansen, Harald S.
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- Synthesis and molecular targets of N-13-hydroxy-octadienoyl-ethanolamine, a novel endogenous bioactive 15-lipoxygenase-derived metabolite of N-linoleoyl-ethanolamine found in the skin and saliva
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N-Arachidonoyl-ethanolamine (AEA) is an endocannabinoid (eCB) and endogenous lipid mimicking many of the effects of Δ9-tetrahydrocannabinol, notably on brain functions, appetite, pain and inflammation. The eCBs and eCB-like compounds contain fatty acids, the main classes being the monoacylglycerols and the N-acyl-ethanolamines (NAEs). Thus, each long chain fatty acid likely exists under the form of a monoacylglycerol and NAE, as it is the case for arachidonic acid (AA) and linoleic acid (LA). Following their biosynthesis, AA and AEA can be further metabolized into additional eicosanoids, notably by the 15-lipoxygenase pathway. Thus, we postulated that NAEs possessing a 1Z,4Z-pentadiene motif, near their omega end, would be transformed into their 15-lipoxygenase metabolites. As a proof of concept, we investigated N-linoleoyl-ethanolamine (LAE). We successfully synthesized LEA and LEA-d4 as well as their 15-lipoxygenase-derived derivatives, namely 13-hydroxy-9Z,11E-octadecadienoyl-N-ethanolamine (13-HODE-EA) and 13-HODE-EA-d4, using Novozyme 435 immobilized on acrylic resin and soybean lipoxygenase respectively. We also show that both human 15-lipoxygenase-1 and -2 can biosynthesize 13-HODE-EA. Co-incubation of LEA and LA with either human 15-lipoxygenase led to the biosynthesis of 13-HODE-EA and 13-HODE in a ratio equal to or greater than 3:1, indicating that LEA is preferred to LA by these enzymes. Finally, we show that 13-HODE-EA is found in human saliva and skin and is a weak although selective TRPV1 agonist. The full biological importance of 13-HODE-EA remains to be explored.
- Tinto, Francesco,Archambault, Anne-Sophie,Dumais, élizabeth,Rakotoarivelo, Volatiana,Kostrzewa, Magdalena,Martin, Cyril,Plante, Pier-Luc,Desjardins, Yves,Simard, Mélissa,Pouliot, Roxane,De Petrocellis, Luciano,Ligresti, Alessia,Di Marzo, Vincenzo,Flamand, Nicolas
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- A Convenient Protocol for the Synthesis of Fatty Acid Amides
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Several classes of biologically occurring fatty acid amides have been reported from mammalian and plant sources. Many amides conjugated with fatty acids of mammalian origin exhibit specific activation of individual receptors. Their potential as pharmacological tools or as lead compounds towards the development of novel therapeutics is of great interest. Hence, access to such amides by a practical, high-yielding and scalable protocol without affecting the geometry or position of sensitive functionalities is needed. A protocol that meets all these requirements involves activation of the corresponding acid with carbonyl diimidazole (CDI) followed by reaction with the desired amine or its hydrochloride. More than fifty compounds have been prepared in generally high yields.
- Johansson, Silje J. R.,Johannessen, Tonje,Ellefsen, Christiane F.,Ristun, Mali S.,Antonsen, Simen,Hansen, Trond V.,Stenstrom, Yngve,Nolsoe, Jens M. J.
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supporting information
p. 213 - 217
(2019/01/14)
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- Scalable synthesis of oleoyl ethanolamide by chemical amidation in a mixed solvent
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Oleoyl ethanolamide is a lipid mediator that exhibits biological activity in animal and cell models. In this study, an effective process is described to synthesize oleoyl ethanolamide by chemical amidation with native oil used as an acyl donor in the presence of sodium methoxide. Reaction conditions were optimized. When the amidation reaction was conducted in a mixed solvent, by reacting 2 mmol high oleic sunflower oil and 20 mmol ethanolamine in the presence of 1.5 % sodium methoxide with agitation, >90 % fatty acid ethanolamide was formed after 3 h of reaction time. The fatty acid ethanolamide product was purified by a two-step crystallization process to prepare oleoyl ethanolamide. Highly pure oleoyl ethanolamide was obtained in a 70.3 % molar yield. The novelty of the work is the use of native oil as acyl donor and the mixed solvent used as the reaction media. The use of native oil avoids the formation of ion pairs with ethanolamine that can occur in other synthesis routes.
- Wang, Xiaosan,Han, Zhengyang,Chen, Yang,Jin, Qingzhe,Wang, Xingguo
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p. 125 - 131
(2016/01/12)
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- Optimized synthesis and characterization of N-acylethanolamines and O-acylethanolamines, important family of lipid-signalling molecules
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The endocannabinoid anandamide (N-arachidonoylethanolamine, AEA), a physiologically occurring bioactive compound on CB1 and CB2 receptors, has multiple physiological functions. Since the discovery of AEA additional non-cannabinoid endogenous compounds such as N-palmitoylethanolamine (PEA), and N-oleoylethanolamine (OEA) have been identified from mammalian tissues. Virodhamine (O-arachidonoylethanolamine, VA) is the only identified new member of the endocannabinoid family that is characterised by an ester linkage between acylic acid and ethanolamine instead of the amide linkage found in AEA and others non-cannabinoid N-acylethanolamines. It has been reported, as a cautionary note for lipid analyses, that VA can be produced nonenzymatically from AEA (and vice versa) as consequence of O,N-acyl migrations. O,N-acyl migrations are well documented in synthetic organic chemistry literature, but are not well described or recognized with regard to methods in lipid isolation or lipid enzyme studies. We here report an economical and effective protocol for large scale synthesis and characterization of some N- and O-acylethanolamines that could be useful as reference standards in order to investigate their possible formation in biological membranes, with potentially interesting biological properties.
- Ottria, Roberta,Casati, Silvana,Ciuffreda, Pierangela
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p. 705 - 711
(2013/01/15)
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- Evaluation of endogenous fatty acid amides and their synthetic analogues as potential anti-inflammatory leads
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A series of endogenous fatty acid amides and their analogues (1-78) were prepared, and their inhibitory effects on pro-inflammatory mediators (NO, IL-1β, IL-6, and TNF-α) in LPS-activated RAW264.7 cells were evaluated. Their inhibitory activity on the pro-inflammatory chemokine MDC in IFN-γ-activated HaCaT cells was also examined. The results showed that the activity is strongly dependent on the nature of the fatty acid part of the molecules. As expected, the amides derived from enone fatty acids showed significant activity and were more active than those derived from other types of fatty acids. A variation of the amine headgroup also altered bioactivity profile remarkably, possibly by modulating cell permeability. Regarding the amine part of the molecules, N-acyl dopamines exhibited the most potent activity (IC50 ～2 μM). This is the first report of the inhibitory activity of endogenous fatty acid amides and their analogues on the production of nitric oxide, cytokines (IL-1β, IL-6, and TNF-α) and the chemokine MDC. This study suggests that the enone fatty acid-derived amides (such as N-acyl ethanolamines and N-acyl amino acids) and N-acyl dopamines may be potential anti-inflammatory leads.
- Dang, Hung The,Kang, Gyeoung Jin,Yoo, Eun Sook,Hong, Jongki,Choi, Jae Sue,Kim, Hyung Sik,Chung, Hae Young,Jung, Jee H.
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experimental part
p. 1520 - 1527
(2011/03/23)
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- Radiosynthesis, in vitro and in vivo evaluation of 123I-labeled anandamide analogues for mapping brain FAAH
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Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids, including anandamide. This paper is the first report of the synthesis, [123I]-labeling and in vitro and in vivo evaluation of anandamide analogues as potential metabolic trapping radioligands for in vivo evaluation of brain FAAH. N-(2-Iodoethyl)linoleoylamide (2) and N-(2-iodoethyl)arachidonylamide (4) were synthesized with good yields (75% and 86%, respectively) in a two steps procedure starting from their respective acids. In vitro analyses, performed using recombinant rat FAAH and [3H]-anandamide, demonstrated interaction of 2 and 4 with FAAH (IC50 values of 5.78 μM and 3.14 μM, respectively). [123I]-2 and [123I]-4 were synthesized with radiochemical yields of 21% and 12%, respectively, and radiochemical purities were >90%. Biodistribution studies in mice demonstrated brain uptake for both tracers (maximum values of 1.23%ID/g at 3 min pi for [123I]-2 and 0.58%ID/g at 10 min pi for [123I]-4). However, stability studies demonstrated the sensitivity of both tracers to dehalogenation.
- wyffels, Leonie,De Bruyne, Sylvie,Blanckaert, Peter,Lambert, Didier M.,De Vos, Filip
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experimental part
p. 49 - 56
(2011/02/25)
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- Selective synthesis of unsaturated N-acylethanolamines by lipase-catalyzed N-acylation of ethanolamine with unsaturated fatty acids
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The selective synthesis of unsaturated N-acylethanolamines 1b-6b by lipase-catalyzed direct condensation between unsaturated fatty acids 1a-6a and ethanolamine is reported. Reactions were carried out in hexane at 40 °C, in the presence of Candida antarctica Lipase B as the catalyst, to give the corresponding amides 1b-6b with yields ranging from 80 to 88%.
- Plastina, Pierluigi,Meijerink, Jocelijn,Vincken, Jean-Paul,Gruppen, Harry,Witkamp, Renger,Gabriele, Bartolo
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experimental part
p. 444 - 447
(2010/04/23)
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- Synthesis, in vitro and in vivo evaluation, and radiolabeling of aryl anandamide analogues as candidate radioligands for in vivo imaging of fatty acid amide hydrolase in the brain
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Fatty acid amide hydrolyase (FAAH) is one of the main enzymes responsible for terminating the signaling of endocannabinoids in the brain. Imaging FAAH in vivo using PET or SPECT is important to deeper understanding of its role in neuropsychiatric disorders. However, at present, no radioligand is available for mapping the enzyme in vivo. Here, we synthesized 18 aryl analogues of anandamide, FAAH's endogenous substrate, and in vitro evaluated their potential as metabolic trapping tracers. Interaction studies with recombinant FAAH revealed good to very good interaction of the methoxy substituted aryl anandamide analogues 17, 18, 19, and 20 with FAAH and they were identified as competing substrates. Compounds 17 and 18 did not display significant binding to CB1 and CB2 cannabinoid receptors and stand out as potential candidate metabolic trapping tracers. They were successfully labeled with 11C in good yields and high radiochemical purity and displayed brain uptake in C57BL/6J mice. Radioligands [11C]-17 and [ 11C]-18 merit further investigation in vivo.
- Wyffels, Leonie,Muccioli, Giulio G.,De Bruyne, Sylvie,Moerman, Lieselotte,Sambre, Johan,Lambert, Didier M.,De Vos, Filip
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supporting information; experimental part
p. 4613 - 4622
(2010/02/28)
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- Novel analogues of arachidonylethanolamide (anandamide): Affinities for the CB1 and CB2 cannabinoid receptors and metabolic stability
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Several analogues of the endogenous cannabinoid receptor ligand arachidonylethanolamide (anandamide) were synthesized and evaluated in order to study (a) the structural requirements for high-affinity binding to the CB1 and CB2 cannabinoid receptors and (b) their hydrolytic stability toward anandamide amidase. The series reported here was aimed at exploring structure-activity relationships (SAR) primarily with regard to stereoelectronic requirements of ethanolamido headgroup for interaction with the cannabinoid receptor active site. Receptor affinities, reported as K(i) values, were obtained by a standard receptor binding assay using [3H]CP- 55,940 as the radioligand, while stability toward the amidase was evaluated by comparing the K(i) of each analogue in the presence and absence of phenylmethanesulfonyl fluoride (PMSF), a serine protease blocker and inhibitor of anandamide amidase. Introduction of a methyl group in the 1'- and 2'-positions or substitution of the ethanolamido headgroup with a butylamido group gave analogues with vastly improved biochemical stability. This is accomplished in some cases with increased receptor affinity. Conversely, oxazolyl and methyloxazolyl headgroups led to low-affinity analogues. Substitution of the hydroxyl group with electronegative substituents such as fluoro, chloro, allyl, and propargyl groups significantly increased receptor affinity but did not influence the biochemical stability. The 2'-chloro analogue of anandamide was found to have the highest affinity for CB1. Additionally, reversing the positions of the carbonyl and NH in the amido group produces retro-anandamides possessing considerably higher metabolic stability. Replacement of the arachidonyl tail with oleyl or linoleyl results in analogues with low affinities for both receptors. All of the analogues in this study showed high selectivity for the CB1 receptor over the peripheral CB2 receptor. The most potent analogues were tested for their ability to stimulate the binding of [35S]GTPγS to G- proteins and were shown to be potent cannabimimetic agonists. The results are discussed in terms of pharmacophoric features affecting receptor affinity and enzymatic stability.
- Lin, Sonyuan,Khanolkar, Atmaram D.,Fan, Pusheng,Goutopoulos, Andreas,Qin, Ce,Papahadjis, Demetris,Makriyannis, Alexandras
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p. 5353 - 5361
(2007/10/03)
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- Facile synthesis of lysophospholipids containing unsaturated fatty acid chains
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The efficient synthesis of polyunsaturated phospholipids is challenging due to the sensitivity of the unsaturated moiety to the conditions employed in phosphate ester deprotection. We discuss here three independent methods that resolve this issue and enable the synthesis of a series of unsaturated lysophosphatidic acid mimics for the development of a more comprehensive understanding of the structure-activity relationship in this series.
- Hopper, Darrin W.,Catalano, John G.,Macdonald, Timothy L.
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p. 7871 - 7874
(2007/10/03)
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