molecules Review Synthesis, Quantification, and Characterization of Fatty Acid Amides from In Vitro and In Vivo Sources Ruidong Ni, Suzeeta Bhandari, Perry R. Mitchell, Jr., Gabriela Suarez, Neel B. Patel, Kara Lamb, Kirpal S. Bisht * and David J. Merkler * Department of Chemistry, University of South Florida, 4202 E. Fowler Ave., Tampa, FL 33620, USA; [email protected] (R.N.); [email protected] (S.B.); [email protected] (P.R.M.J.); [email protected] (G.S.); [email protected] (N.B.P.); [email protected] (K.L.) * Correspondence: [email protected] (K.S.B.); [email protected] (D.J.M.); Tel.: +1-813-974-0350 (K.S.B.); +1-813-974-3579 (D.J.M.) Abstract: Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented. Keywords: fatty acid amide; liquid chromatography/quadrupole time-of-flight mass spectrometry; anandamide; N18TG2; choroid plexus; Drosophila; Bombyx; Apis; Tribolium Citation: Ni, R.; Bhandari, S.; Mitchell, P.R., Jr.; Suarez, G.; Patel, N.B.; Lamb, K.; Bisht, K.S.; Merkler, D.J. Synthesis, Quantification, and Characterization of Fatty Acid 1. Introduction Amides from In Vitro and In Vivo Fatty acid amides are a family of intriguing, yet structurally simple lipids, R-CO-NH- Sources. Molecules 2021, 26, 2543. R’. The acyl moiety, R-CO-, is derived from the fatty acids listed in most undergraduate https://doi.org/10.3390/molecules biochemistry textbooks and the -NH-R’ moiety is derived from the set of biogenic amines. 26092543 The structural simplicity of the fatty acid amides belies both the importance and diver- sity of this lipid family. Hundreds of different fatty acid amides are possible and, to Academic Editor: Pierangela Ciuffreda date, approximately 90 different fatty acid amides have been identified from living or- ganisms [1]. In vivo, accumulating evidence suggests that the fatty acid amides are cell Received: 5 April 2021 signaling lipids [2–6] and have a technological use as slip additives in plastics [7]. Accepted: 23 April 2021 The biological occurrence of the fatty acid amide bond traces back to the 1880s with Published: 27 April 2021 the first characterization of sphingomyelin by Thudichum [8]. Decades after the work of Thudichum, N-palmitoylethanolamine was isolated from egg yolk [9] and five different Publisher’s Note: MDPI stays neutral primary fatty acid amides were identified in luteal phase plasma [10]. Interest in the fatty with regard to jurisdictional claims in acid amides increased dramatically after the identification of N-arachidonoylethanolamine published maps and institutional affil- (anandamide) as the endogenous ligand of the CB receptor found in the mammalian iations. 1 brain [11]. Other key discoveries cementing the biological importance of the fatty acid amides were the demonstration that N-(17-hydroxylinolenoyl)-L-glutamine (volicitin) as an elicitor of plant volatiles produced in insects [12] and the characterization of oleamide as a regulator of the sleep/wake cycle found in the mammalian brain [13]. Endocannabinoids Copyright: © 2021 by the authors. are endogenous lipid-based ligands that bind to the CB1 and CB2 receptors [4,5]. The fatty Licensee MDPI, Basel, Switzerland. acid amides are, thus, endocannbinoid-like or endocannbinoid related by virtue of their This article is an open access article structural similarity to anandamide. distributed under the terms and Our interest in the fatty acid amides stemmed from the discovery that peptidylglycine conditions of the Creative Commons α-amidating monooxygenase (PAM) catalyzes the oxidation of N-fatty acylglycines to Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ primary fatty acid amides (PFAMs) [14–16]. Our initial focus on the N-fatty acylglycines 4.0/). and the PFAMs lead to broader interest in the fatty acid amide family and the identification Molecules 2021, 26, 2543. https://doi.org/10.3390/molecules26092543 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 14 Molecules 2021, 26, 2543 2 of 13 and the PFAMs lead to broader interest in the fatty acid amide family and the identifica- tion of N-acyltransferases responsible for their biosynthesis [17–19]. One aspect of our broaderof N-acyltransferases interest in the responsible fatty acid foramide their family biosynthesis were studies [17–19 ].to One isolate aspect and of quantify our broader the fattyinterest acid in amides the fatty produced acid amide in model family organisms, were studies called to isolate the fatty and acid quantify amidome. the fatty We syn- acid thesizedamidesproduced specific fatty in model acid amides organisms, as standards called the for fatty the liquid acid amidome. chromatography/quadru- We synthesized polespecific time fatty-of-flight acid amides mass spectrometry as standards (LC/QTOF for the liquid-MS) chromatography/quadrupole method to define the fatty acid time-of- am- idomeflight mass [18,20]. spectrometry In this review, (LC/QTOF-MS) we first describe method the to synthesis define the and fatty characterization acid amidome of [18 spe-,20]. cificIn this fatty review, acid amides we first and describe then summarize the synthesis our andresults characterization on the characterization of specific of fatty the fatty acid acidamides amidome and then from summarize cultured ourmammalian results on cells the [15] characterization and insects [18 of– the21]. fatty The novelty acid amidome of this from cultured mammalian cells [15] and insects [18–21]. The novelty of this article relative article relative to other reviews on the endocannbinoids [1–6] is a broad focus on the en- to other reviews on the endocannbinoids [1–6] is a broad focus on the endocannbinoid- docannbinoid-related fatty acid amides, coverage of the fatty acid amides identified in related fatty acid amides, coverage of the fatty acid amides identified in insects, and the insects, and the methodologies for chemical synthesis of the standards that were used in methodologies for chemical synthesis of the standards that were used in the characteriza- the characterization of the fatty acid amidome. tion of the fatty acid amidome. 2. Results Results and and Discussion Discussion 2.1. Synthesis Synthesis of of the the Fatty Acid Amides The synthetic ro routesutes and structures of the fatty acid amides prepared in this study are shown in in Figure Figure 11.. The The condensation condensation of of the the corresponding corresponding fatty fatty acid acid chloride chloride with with 2- aminoethanol2-aminoethanol or or glycine glycine in in the the presence presence of of triethylamine triethylamine or or NaOH, NaOH, respectively, respectively, gave the desired fat fattyty acid amide ( FAFA-1-1, 2,, andand 44)) inin goodgood toto excellentexcellent yieldyield [[22,23].22,23]. PalmitamidePalmitamide (FAFA-3-3) waswas obtained obtained by by condensation condensation of ofthe the palmitoyl palmitoyl chloride chloride with with ammonia ammonia in the in pres- the encepresence of triethylamine of triethylamine [24]. [24]. Figure 1. The synthetic strategy for the fatty acid amides. 2.2. Structural Structural Analysis Analysis of the Synthetically Prepared Fatty Acid Amides The molecular structures structures of of the fatty acid amides FAFA-1-1, 2, 3 and 4 were confirmedconfirmed spectroscopically ( (11HH and and 13C13-C-NMR,NMR, and and MS; MS; Supplementary Supplementary Materials Materials Figures Figures S1–S8) S1–S8) and uponand upon comparison comparison to reported to reported literature literature values. values. All the obtained All the obtained fatty acid fatty amides acid 1– amides4 were white1–4 were solids. white In solids.the 1H- In and the 131CH--NMR and 13spectra,C-NMR alkenic spectra, (=C alkenicH) hydrogens (=CH) hydrogens in FA-1 and in FA-1FA 2 appearedand FA 2 appearedas a multiplet as a at multiplet 5.3 ppm at and 5.3 the ppm alkenic and the (=C alkenic) carbons (=C were) carbons observed were at observed 129 and 130at 129 ppm, and respectively. 130 ppm, respectively. The NHCH2 The hydrogens NHCH2 inhydrogens FA-1 were in observedFA-1 were as observed a triplet asof a doublettriplet of at a 3.4 doublet ppm at(J = 3.4 4.8 ppm and (J5.2 = 4.8Hz), and showing 5.2 Hz), coupling showing of couplingthe NH and of the the NH hydrogens and the ofhydrogens the hydroxymethylene of the hydroxymethylene group. The NHC group.H2 hydrogens The NHCH in2 hydrogensFA-2 and FA in-4FA-2 appearedand FA-4 as a doubletappeared (J as= 6 a Hz) doublet at 3.7 (J ppm,= 6 Hz) due at to 3.7 its ppm, coupling due towith its the coupling NH hydrogen. with the NHThe hydrogen.FA-2 and The FA-2 and FA-4, in the 13C-NMR, showed two resonances corresponding to the amide Molecules 2021, 26, 2543 3 of 13 and carboxylic acid carbonyls at 170 and 171 ppm, for FA-1 and FA-3, the single amide carbonyl resonance was observed at ~174 ppm. Each of the fatty acid amide was also subjected to the high-resolution mass spectrometry, and the exact mass of the compounds was determined to be within acceptable instrumental error. 2.3. The Fatty Acid Amidome from Mouse N18TG2 Cells and Sheep Choroid Plexus (SCP) Cells Our initial interest in defining the fatty acid amidome was narrowly focused on the PAM-mediated conversion of N-fatty acylglycines to the PFAMs. To this end, our first model system was mouse neuroblastoma N18TG2 cells, cells known to produce oleamide [25] and other fatty acid amides [26].