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Hydroxy fatty acids (HFAs) constitute a class of lipids, distinguished by the presence of a hydroxyl on a long aliphatic chain. This study aims to expand our insights into HFA bioactivities, while also introducing new methods for asymmetrically synthesizing unsaturated and saturated HFAs. Simultaneously, a procedure previously established by us was adapted to generate new HFA regioisomers. An organocatalytic step was employed for the synthesis of chiral terminal epoxides, which either by alkynylation or by Grignard reagents resulted in unsaturated or saturated chiral secondary alcohols and, ultimately, HFAs. 7-( S )-Hydroxyoleic acid (7SHOA), 7-( S )-hydroxypalmitoleic acid (7SHPOA) and 7-( R )- and ( S )-hydroxymargaric acids (7HMAs) were synthesized for the first time and, together with regioisomers of ( R )- and ( S )-hydroxypalmitic acids (HPAs) and hydroxystearic acids (HSAs), whose biological activity has not been tested so far, were studied for their antiproliferative activities. The unsaturation of the long chain, as well as an odd-numbered (C17) fatty acid chain, led to reduced activity, while the new 6-( S )-HPA regioisomer was identified as exhibiting potent antiproliferative activity in A549 cells. 6SHPA induced acetylation of histone 3 in A549 cells, without affecting acetylated α-tubulin levels, suggesting the selective inhibition of histone deacetylase (HDAC) class I enzymes, and was found to inhibit signal transducer and activator of transcription 3 (STAT3) expression.

The development of novel agents to combat COVID-19 is of high importance. SARS-CoV-2 main protease (M pro ) is a highly attractive target for the development of novel antivirals and a variety of inhibitors have already been developed. Accumulating evidence on the pathobiology of COVID-19 has shown that lipids and lipid metabolizing enzymes are critically involved in the severity of the infection. The purpose of the present study was to identify an inhibitor able to simultaneously inhibit both SARS-CoV-2 M pro and phospholipase A 2 (PLA 2 ), an enzyme which plays a significant role in inflammatory diseases. Evaluating several PLA 2 inhibitors, we demonstrate that the previously known potent inhibitor of Group IIA secretory PLA 2 , GK241, may also weakly inhibit SARS-CoV-2 M pro . Molecular mechanics docking and molecular dynamics calculations shed light on the interactions between GK241 and SARS-CoV-2 M pro . 2-Oxoamide GK241 may represent a lead molecular structure for the development of dual PLA 2 and SARS-CoV-2 M pro inhibitors.

The quest for novel agents to regulate the generation of prostaglandin E 2 (PGE 2 ) is of high importance because this eicosanoid is a key player in inflammatory diseases. We synthesized a series of N -acylated and N -alkylated 2-aminobenzothiazoles and related heterocycles (benzoxazoles and benzimidazoles) and evaluated their ability to suppress the cytokine-stimulated generation of PGE 2 in rat mesangial cells. 2-Aminobenzothiazoles, either acylated by the 3-(naphthalen-2-yl)propanoyl moiety (GK510) or N -alkylated by a chain carrying a naphthalene (GK543) or a phenyl moiety (GK562) at a distance of three carbon atoms, stand out in inhibiting PGE 2 generation, with EC 50 values ranging from 118 nM to 177 nM. Both GK510 and GK543 exhibit in vivo anti-inflammatory activity greater than that of indomethacin. Thus, N -acylated or N -alkylated 2-aminobenzothiazoles are novel leads for the regulation of PGE 2 formation.

Unsaturated nitro fatty acids (NO 2 -FAs) constitute a category of molecules that may be formed endogenously by the reaction of unsaturated fatty acids (UFAs) with secondary species of nitrogen monoxide and nitrite anions. The warhead of NO 2 -FAs is a nitroalkene moiety, which is a potent Michael acceptor and can undergo nucleophilic attack from thiol groups of biologically relevant proteins, showcasing the value of these molecules regarding their therapeutic potential against many diseases. In general, NO 2 -FAs inhibit nuclear factorκ-B (NF-κB), and simultaneously they activate nuclear factor (erythroid derived)-like 2 (Nrf2), which activates an antioxidant signaling pathway. NO 2 -FAs can be synthesized not only endogenously in the organism, but in a synthetic laboratory as well, either by a step-by-step synthesis or by a direct nitration of UFAs. The step-by-step synthesis requires specific precursor compounds and is in position to afford the desired NO 2 -FAs with a certain position of the nitro group. On the contrary, the direct nitration of UFAs is not a selective methodology; thus, it affords a mixture of all possible nitro isomers.

Prostaglandin E 2 (PGE 2 ) is a key mediator of inflammation, and consequently huge efforts have been devoted to the development of novel agents able to regulate its formation. In this work, we present the synthesis of various α-ketoheterocycles and a study of their ability to inhibit the formation of PGE 2 at a cellular level. A series of α-ketobenzothiazoles, α-ketobenzoxazoles, α-ketobenzimidazoles, and α-keto-1,2,4-oxadiazoles were synthesized and chemically characterized. Evaluation of their ability to suppress the generation of PGE 2 in interleukin-1β plus forskolin-stimulated mesangial cells led to the identification of one α-ketobenzothiazole (GK181) and one α-ketobenzoxazole (GK491), which are able to suppress the PGE 2 generation at a nanomolar level.

Oxidized saturated fatty acids, containing a hydroxyl or an oxo functionality, have attracted little attention so far. Recent studies have shown that saturated hydroxy fatty acids, which exhibit cancer cell growth inhibition and may suppress β-cell apoptosis, are present in milk. Herein, we present the application of a liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for the detection and quantification of various saturated oxo fatty acids (SOFAs) previously unrecognized in milk. This robust and rapid analytical method, which involves simple sample preparation and a single 10-min run, revealed the presence of families of oxostearic acids (OSAs) and oxopalmitic acids (OPAs) in milk. 8OSA, 9OSA, 7OSA, 10OSA and 10OPA were found to be the most abundant SOFAs in both cow and goat milk. Higher contents of SOFAs were found in cow milk in comparison to goat milk. Together with SOFAs, ricinoleic acid, which is isobaric to OSA, was detected and quantified in all milk samples, following a "suspect" HRMS analysis approach. This unique natural fatty acid, which is the main component (>90%) of castor oil triglycerides, was estimated at mean content values of 534.3 ± 6.0 μg/mL and 460 ± 8.1 μg/mL in cow and goat milk samples, respectively.

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the direct determination of various saturated hydroxy fatty acids (HFAs) in milk was developed for the first time. The method involves mild sample preparation conditions, avoids time-consuming derivatization procedures, and permits the simultaneous determination of 19 free HFAs in a single 10-min run. This method was validated and applied in 17 cow milk and 12 goat milk samples. This work revealed the existence of various previously unrecognized hydroxylated positional isomers of palmitic acid and stearic acid in both cow and goat milk, expanding our knowledge on the lipidome of milk. The most abundant free HFAs in cow milk were proven to be 7-hydroxystearic acid (7HSA) and 10-hydroxystearic acid (10HSA) (mean content values of 175.1 ± 3.4 µg/mL and 72.4 ± 6.1 µg/mL in fresh milk, respectively). The contents of 7HSA in cow milk seem to be substantially higher than those in goat milk.

Adrenic acid (AA), the 2-carbon elongation product of arachidonic acid, is present at significant levels in membrane phospholipids of mouse peritoneal macrophages. Despite its abundance and structural similarity to arachidonic acid, very little is known about the molecular mechanisms governing adrenic acid mobilization in cells of the innate immune system. This contrasts with the wide availability of data on arachidonic acid mobilization. In this work, we used mass-spectrometry-based lipidomic procedures to define the profiles of macrophage phospholipids that contain adrenic acid and their behavior during receptor activation. We identified the phospholipid sources from which adrenic acid is mobilized, and compared the data with arachidonic acid mobilization. Taking advantage of the use of selective inhibitors, we also showed that cytosolic group IVA phospholipase A 2 is involved in the release of both adrenic and arachidonic acids. Importantly, calcium independent group VIA phospholipase A 2 spared arachidonate-containing phospholipids and hydrolyzed only those that contain adrenic acid. These results identify separate mechanisms for regulating the utilization of adrenic and arachidonic acids, and suggest that the two fatty acids may serve non-redundant functions in cells.

The determination of free fatty acids (FFAs) in milk is of importance for quality control, legislative purposes, authentication and product development. We present herein a liquid chromatography-high resolution mass spectrometry method for the direct determination of FFAs in milk. The method involves mild sample preparation, avoids time-consuming derivatization and allows the direct quantification of twenty-two FFAs in a 10-min single run. It was validated and applied in thirteen cow milk and seven goat milk samples. Saturated fatty acids C16:0, C18:0 and unsaturated C18:1 (n-9) were found to be the major components of milk FFAs at concentrations of 33.1 ± 8.2 μg/mL, 16.5 ± 5.3 μg/mL and 14.8 ± 3.8 μg/mL, respectively, in cow milk and at concentrations of 22.8 ± 1.8 μg/mL, 12.7 ± 2.8 μg/mL and 13.3 ± 0.3 μg/mL, respectively, in goat milk. Other saturated and unsaturated FFAs were found in significantly lower quantities. Saturated fatty acids C6:0, C8:0 and C10:0 were found in higher quantities in goat milk than in cow milk. The levels of the important (for human health) odd-chain FFAs C15:0 and C17:0 were estimated in cow and goat milk.

2-Oxoesters constitute an important class of potent and selective inhibitors of human cytosolic phospholipase A 2 (GIVA cPLA 2 ) combining an aromatic scaffold or a long aliphatic chain with a short aliphatic chain containing a free carboxylic acid. Although highly potent 2-oxoester inhibitors of GIVA cPLA 2 have been developed, their rapid degradation in human plasma limits their pharmaceutical utility. In an effort to address this problem, we designed and synthesized two new 2-oxoesters introducing a methyl group either on the α-carbon to the oxoester functionality or on the carbon carrying the ester oxygen. We studied the in vitro plasma stability of both derivatives and their in vitro inhibitory activity on GIVA cPLA 2 . Both derivatives exhibited higher plasma stability in comparison with the unsubstituted compound and both derivatives inhibited GIVA cPLA 2 , however to different degrees. The 2-oxoester containing a methyl group on the α-carbon atom to the oxoester functionality exhibits enhancement of the metabolic stability and retains considerable inhibitory potency., Competing Interests: The authors have declared no conflict of interest.

The lipidome of royal jelly (RJ) consists of medium-chained (8-12 carbon atoms) free fatty acids. We present herein a liquid chromatography-high resolution mass spectrometry (HRMS) method that permits the determination of RJ fatty acids and at the same time the detection of suspect fatty acids. The method allows for the direct quantification of seven free fatty acids of RJ, avoiding any derivatization step. It was validated and applied in seven RJ samples, where the major RJ fatty acid trans-10-hydroxy-2-decenoic acid (10-HDA) was found to vary from 0.771 ± 0.08 to 0.928 ± 0.04 g/100 g fresh RJ. Four additional suspect fatty acids were simultaneously detected taking advantage of the HRMS detection.

Availability of free arachidonic acid (AA) constitutes a rate limiting factor for cellular eicosanoid synthesis. AA distributes differentially across membrane phospholipids, which is largely due to the action of coenzyme A-independent transacylase (CoA-IT), an enzyme that moves the fatty acid primarily from diacyl phospholipid species to ether-containing species, particularly the ethanolamine plasmalogens. In this work, we examined the dependence of AA remodeling on plasmalogen content using the murine macrophage cell line RAW264.7 and its plasmalogen-deficient variants RAW.12 and RAW.108. All three strains remodeled AA between phospholipids with similar magnitude and kinetics, thus demonstrating that cellular plasmalogen content does not influence the process. Cell stimulation with yeast-derived zymosan also had no effect on AA remodeling, but incubating the cells in AA-rich media markedly slowed down the process. Further, knockdown of cytosolic-group IVC phospholipase A 2 γ (cPLA 2 γ) by RNA silencing significantly reduced AA remodeling, while inhibition of other major phospholipase A 2 forms such as cytosolic phospholipase A 2 α, calcium-independent phospholipase A 2 β, or secreted phospholipase A 2 had no effect. These results uncover new regulatory features of CoA-IT-mediated transacylation reactions in cellular AA homeostasis and suggest a hitherto unrecognized role for cPLA 2 γ in maintaining membrane phospholipid composition via regulation of AA remodeling.

3-Hydroxy fatty acids have attracted the interest of researchers, since some of them may interact with free fatty acid receptors more effectively than their non-hydroxylated counterparts and their determination in plasma provides diagnostic information regarding mitochondrial deficiency. We present here the development of a convenient and general methodology for the asymmetric synthesis of 3-hydroxy fatty acids. The enantioselective organocatalytic synthesis of terminal epoxides, starting from long chain aldehydes, is the key-step of our methodology, followed by ring opening with vinylmagnesium bromide. Ozonolysis and subsequent oxidation leads to the target products. MacMillan's third generation imidazolidinone organocatalyst has been employed for the epoxide formation, ensuring products in high enantiomeric purity. Furthermore, a route for the incorporation of deuterium on the carbon atom carrying the hydroxy group was developed allowing the synthesis of deuterated derivatives, which may be useful in biological studies and in mass spectrometry studies. In addition, the synthesis of fatty γ-lactones, corresponding to 4-hydroxy fatty acids, was also explored.

Peptide drug discovery may play a key role in the identification of novel medicinal agents. Here, we present the development of novel small peptides able to suppress the production of PGE₂ in mesangial cells. The new compounds were generated by structural alterations applied on GK115, a novel inhibitor of secreted phospholipase A₂, which has been previously shown to reduce PGE₂ synthesis in rat renal mesangial cells. Among the synthesized compounds, the tripeptide derivative 11 exhibited a nice dose-dependent suppression of PGE₂ production, similar to that observed for GK115., Competing Interests: The authors declare no conflict of interest.