Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Synthesis and biological evaluation of 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives as protein kinase inhibitors

The synthesis and biological evaluation of a number of differently substituted 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives are reported. From the inhibition results on a selection of disease-relevant protein kinases [IC₅₀ (μM) DYRK1A = 11; CDK5 = 0.41; GSK-3 = 1.5] we have observed that 3,6-diamino-4-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4) constitutes a potential new and simple lead compound in the search of drugs for the treatment of Alzheimer’s disease.     

High pressure liquid chromatography methods for separation of omega- and (omega-1)-hydroxy fatty acids: their applications to microsomal fatty acid omega-oxidation

Fatty acids (C12-C18) and their omega- and (omega-1)-hydroxy derivatives, when converted to p-bromophenacyl (PBP) esters, can be completely separated from one another by high pressure liquid chromatography (HPLC) on a silicic acid column using 0.5% (v/v) isopropanol in n-hexane. In this system, fatty acid PBP esters are eluted at the solvent front, whereas the retention times of the omega- and (omega-1)-hydroxy derivatives are 14-20 and 24-29 min, respectively. The PBP esters can also be separated by reverse phase HPLC on a muBondapak C18 column, a method which has been developed by Fan et al. (Fan, L. L., Masters, B. S. S., and Prough, R. A. (1976) Anal. Biochem. 71, 265-272) for separation of methyl esters of fatty acids and their omega- and (omega-1)-hydroxy derivatives. In the latter method, however, the retention times of omega- and (omega-1)-hydroxy derivatives are only about 2 min apart and an increase in the solvent polarity is needed for elution of the esters of unmodified fatty acids. Fatty acid PBP esters, however, can be obtained as independent peaks which are not disturbed by the solvent front. An application of the former method to measure fatty acid omega oxidation by liver microsomes and by a reconstituted monooxygenase system containing purified cytochrome P-450 is described.     

6-acyl galactosyl ceramides of pig brain: structure and fatty acid composition

Two glycolipids were isolated from pig brain and were shown to be the fatty acid esters of kerasin and cerebron in which the second fatty acid moiety is attached to the 6-position of the galactose. The point of attachment was shown in two ways: by permethylation and by cleavage with periodate. Methanolysis of the permethylated cerebroside esters yielded O-methyl sphingosines, methyl esters of nonhydroxy or 2-methoxy acids, and methyl 2,3,4-trimethyl galactoside. Cleavage of the cerebron ester with periodate, followed by treatment with sodium borohydride and dilute HCl, yielded ceramide plus 1-monoglyceride. The ester-linked fatty acids were primarily 16:0, 18:0, and 18:1, while the amide-linked fatty acids showed the wide assortment of chain lengths typical of brain cerebrosides. The methylation step, with silver oxide and methyl iodide, yielded two derivatives with the cerebroside esters, but the structural explanation for the difference was not elucidated. The galactose in the cerebron ester was shown to exist in the beta-pyranoside form.     

Stereospecific induction of starfish oocyte maturation by (8R)-hydroxyeicosatetraenoic acid

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine. This induction of meiotic divisions can be triggered also by four fatty acids: 5,8,11-20:3; 5,8,11,14-20:4 (arachidonic acid); 6,9,12,15-20:4; 5,8,11,14,17-20:5, all other fatty acids being completely inactive. This maturation triggered by eicosanoids occurs in the micromolar range and is facilitated by the presence of calcium. A variety of arachidonic acid derivatives (esters, epoxides, etc.) and metabolites (cyclooxygenase and lipoxygenase products) has been tested; the biological activity is restricted to 8-hydroxyeicosatetraenoic acid (8-HETE), other mono- and poly-HETEs being completely inactive. Maturation triggered by 8-HETE occurs around 10 nM and is insensitive to the presence of calcium. 8-HETE methyl ester and 8-hydroperoxyeicosatetraenoic acid are able to induce maturation at higher concentrations. Both (8S) and (8R) stereoisomers have been tested; the biological activity is strictly restricted to the (8R) isomer. 8-HETE triggers a complete maturation, i.e. maturation-promoting factor appearance, germinal vesicle breakdown, emission of the polar bodies, and formation of a female pronucleus. (8R)-HETE, but not (8S)-HETE, triggers the typical decrease in cyclic AMP concentration induced by 1-methyladenine and the burst of protein phosphorylation associated with maturation. Starfish oocytes oxidize exogenous arachidonic acid into 8-HETE and other HETEs. 8-HETE was identified, after high pressure liquid chromatography purification, by gas chromatography mass spectrometry. Furthermore, it was found that the starfish oocytes only produce the (8R)-HETE isomer. This highly stereospecific induction of oocyte maturation by (8R)-HETE suggests that this fatty acid, or a very closely related fatty acid, may play a role in the transduction of the 1-methyladenine message at the plasma membrane level.     

2,5:3,4-Dianhydro-1,6-dideoxy-6-(trimethylamino)-d-allitol and -d-galactitol

A new, four-step synthesis of 2,5:3,6-dianhydro-1-deoxy-d-glucitol 16 was worked out, starting from 1,6-dibromo-1,6-dideoxy-d-mannitol. Compound 16 was converted into different 4-O-acyl derivatives, the 3,6-anhydro rings of which where opened with hydrogen bromide, yielding the corresponding 6-bromo compounds. These were converted, via the 6-azides, into the 6-(dimethylamino) derivatives, the sulfonic esters of which gave, on treatment with base, the 2,5:3,4-dianhydro-d-allitol and -d-galactitol derivatives. These were converted with methyl iodide into the corresponding quaternary salts. On biological testing, only the d-allitol derivative showed weak, muscarine-like activity.     

Isolation and taxonomic and catabolic characterization of a 3,6-dimethylphenanthrene-utilizing strain of Sphingomonas sp

A bacterial strain capable of utilizing 3,6-dimethylphenanthrene (3,6-DMP) as its sole source of carbon and energy was isolated from a creosote-contaminated soil. The isolate was identified as a strain of Sphingomonas sp. and was designated strain JS1. Utilization of 3,6-DMP was demonstrated by an increase in bacterial biomass concomitant with a decrease in 3,6-DMP in a liquid mineral medium with this compound as its sole source of carbon and energy. Strain JS1 showed a high specificity in the use of the most abundant alkylderivatives of crude oils, such as alkylnaphthalenes and other alkylphenanthrenes, as the sole source of carbon and energy. It can also use several polycyclic aromatic hydrocarbons of three and four rings and their alkylated derivatives as growth substrates or transform them. The identification of several intermediate metabolites points to extensive metabolic activity, including the following: (i) aromatic ring oxidation and cleavage, (ii) methyl group oxidations, and (iii) methylenic oxidations. The metabolic actions of Sphingomonas sp. JS1 on the aromatic fraction extracted from a creosote-contaminated soil are also examined.     

Chemical modification of the sugar part of methyl acarviosin: synthesis and inhibitory activities of nine analogues.

Nine analogues of methyl acarviosin (1), the core structure of acarbose and its homologues, the 6-hydroxy-(2), 6-azido-(3), 6-amino- (4), 6-acetamido-(5), 6-methoxy-(6), 6-hydroxy-2-O-methyl-(8), and 6-hydroxy-3-O-methyl derivatives (9), including the 5-methoxycarbonyl analogue (7) and 3,6-anhydro derivative (10) of 2, were synthesized by chemical modification of the sugar part of 2 derived by condensation of methyl 3,4-anhydro-alpha-D-galactopyranoside (17) and 4,7:5,6-di-O-isopropylidenevalienamine (26) or by direct coupling between 26 and the 6-substituted methyl 3,4-anhydro-alpha-D-galactopyranoside derivatives. Compounds 2 and 8 show notable inhibitory activity against yeast alpha-D-glucosidase almost comparable to that of 1. Introduction of a polar substituent at C-6 of 1 decreases the inhibitory activity. Interestingly, inversion of the conformation of the sugar part of 1 by introduction of the 3,6-anhydro bridge elicits almost no effect on the inhibitory activity.     

Synthesis of a cyanoethylidene derivative of 3,6-anhydro-d-galactose and its application as glycosyl donor

Starting from 1,2,4-tri-O-acetyl-3,6-anhydro-alpha-d-galactopyranose, 4-O-acetyl-3,6-anhydro-1,2-O-(1-cyanoethylidene)-alpha-d-galactopyranose (7) was synthesized by treatment with cyanotrimethylsilane. Additionally, 3,4-di-O-acetyl-1,2-O-(1-cyanoethylidene)-6-O-tosyl-alpha-d-galactopyranose was prepared from the corresponding bromide and both cyanoethylidene derivatives were used as donors in glycosylation reactions. The coupling with benzyl 2,4,6-tri-O-acetyl-3-O-trityl-beta-d-galactopyranoside provided exclusively the beta-linked disaccharides in approximately 30% yield. The more reactive methyl 2,3-O-isopropylidene-4-O-trityl-alpha-l-rhamnopyranoside gave with donors 3 and 7 the corresponding disaccharides in nearly 60% yield. Furthermore, the synthesis of 3,6-anhydro-4-O-trityl-1,2-O-[1-(endo-cyano)ethylidene]-alpha-d-galactopyranose, which can be used as a monomer for polycondensation reaction is described.     

Isolation of 2-methyl branched unsaturated very long fatty acids from marine sponge Halichondria panicea and identification of them by GC-MS and NMR

In order to study the biomarker fatty acids of symbionts in the marine sponge Halichondria panicea, purification and structural identification of two new 2-methyl branched monoenoic very long fatty acids (2-Me-24:1 n-7 and 2-Me-26:1 n-9) were performed for the first time. These acids amounted to 7.1% of total sponge FAs, but our attempts to determine their structures by one-step GC-MS analysis were unsuccessful because of low yields of the correspondent N-acyl pyrrolidide derivatives. Silver-ion thin-layer chromatography isolated enriched fractions of monoenoic fatty acids extracted from the sponge. Further purification of unknown fatty acid methyl esters was carried out by reversed-phase high-performance liquid chromatography. Determination of the chain length, degree and position of unsaturations was achieved by gas chromatography-mass spectrometry on methyl esters and dimethyldisulfide adducts. Structures, position of methyl substitution, and double bonds cis isomery were confirmed by 1H nuclear magnetic resonance.     

Synthesis and NMR spectroscopy of nine stereoisomeric 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids

Derivatives of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids are essential constituents of some bacterial polysaccharides and glycoproteins. In order to establish reliably the configuration of the natural sugars, nine stereoisomeric 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids were synthesized, including di-N-acetyl-legionaminic and -pseudaminic acids (the D-glycero-D-galacto and L-glycero-L-manno isomers, respectively) and their isomers at C-4, C-5, C-7, and C-8 having the L-glycero-D-galacto, D-glycero-D-talo, L-glycero-D-talo, D-glycero-L-altro, L-glycero-L-altro, D-glycero-L-manno, and L-glycero-L-gluco configurations. Synthesis was performed by condensation of 2,4-diacetamido-2,4,6-trideoxy-L-gulose, -D-mannose, -D-talose, and -L-allose with oxalacetic acid under basic conditions, the reaction of the last two precursors being accompanied by epimerisation at C-2. The 1H and 13C NMR data of the synthetic compounds are discussed. Acetylated methyl esters of the C-7 and C-8 isomeric nonulosonic acids were prepared and used for analysis of the side-chain conformation by NMR spectroscopy.     

Carboxymethyl-substituted bifunctional chelators: preparation of aryl isothiocyanate derivatives of 3-(carboxymethyl)-3-azapentanedioic acid, 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic++ + acid, and 1,4,7,10-tetraazacyclododecane-N,N',N",N'-tetraacetic acid for use as protein labels

3-(Carboxymethyl)-3-azapentanedioic acid (NTA), 3,12-bis(carboxymethyl)-6,9-dioxa-3,12-diazatetradecanedioic acid (EGTA), and 1,4,7,10-tetraazacyclododecane-N,N',N",N'-tetraacetic acid (DOTA) structures having a 4-nitrophenyl substituent attached via an alkyl spacer to the methylene carbon atom of one carboxymethyl arm of the chelator were obtained by alkylation of 4-nitrophenylalanine with bromoacetic acid (NTA), by reductive alkylation of 1,8-diamino-3,6-dioxaoctane with (4-nitrophenyl)-pyruvic acid followed by alkylation with bromoacetic acid (EGTA), and by alkylation of the trimethyl ester of 1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid with the methyl ester of alpha-bromo-4-(4-nitrophenyl)pentanoic acid and subsequent saponification (DOTA). The nitrophenyl-substituted chelators were converted to the corresponding amines by hydrogenation then reacted with thiophosgene to give the protein-reactive aryl isothiocyanate derivatives.     

Phenolic acid derivatives with potential anticancer properties--a structure-activity relationship study. Part 1: methyl, propyl and octyl esters of caffeic and gallic acids

The antiproliferative and cytotoxic properties of polyphenolic acid derivatives, structurally related with the natural models caffeic and gallic acids, have been tested in human cervix adenocarcinoma cells (HeLa). Simultaneous structural information was obtained for these compounds through theoretical ab initio methods. This study was conducted for the following esters: methyl caffeate (MC, 1), propyl caffeate (PC, 2), octyl caffeate (OC, 3), methyl gallate (MG, 4), propyl gallate (PG, 5) and octyl gallate (OG, 6). A significant growth-inhibition effect was assessed for some of these compounds, clearly dependent on their structural characteristics. Marked structure-activity relationships (SARs)--namely the number of hydroxyl ring substituents--were found to rule the biological effect of such systems.     

Determination of monoenoic fatty acid double bond position by permanganate-periodate oxidation followed by high-performance liquid chromatography of carboxylic acid phenacyl esters

This investigation was carried out to develop methods for a reverse-phase, high-performance liquid chromatography analysis of the monocarboxylic and dicarboxylic acids produced by permanganate-periodate oxidation of monoenoic fatty acids. Oxidation reactions were performed using [U-14C]oleic acid and [U-14C]oleic acid methyl ester in order to measure reaction yields and product distributions. The 14C-labeled oxidation products consisted of nearly equal amounts of monocarboxylic and dicarboxylic acid (or dicarboxylic acid monomethyl ester), with few side products (yield greater than 98%). Conversion of the carboxylic acids to phenacyl esters proceeded to completion. HPLC of carboxylic acid phenacyl esters was performed using a C18 column with a linear solvent gradient beginning with acetonitrile/water (1/1) and ending with 100% acetonitrile. Excellent resolution was achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid phenacyl esters. Resolution was also achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid monomethyl, monophenacyl esters. The resolution obtained by HPLC demonstrates that, for a wide range of monoenoic fatty acids, both products of a permanganate-periodate oxidation can be identified on a single chromatogram. Free fatty acids and fatty acid methyl esters were analyzed with equal success. Neither the oxidation nor the esterification reaction caused detectable hydrolysis of methyl ester. The method is illustrated for free acids and methyl esters of 14:1 (cis-9), 16:1 (cis-9), 18:1 (cis-6), 18:1 (cis-9), and 18:1 (cis-11).     

Monoepoxy octadecadienoates and monoepoxy octadecatrienoates 1: NMR spectral characterization

Methyl esters of gamma-linolenic acid, alpha-linolenic acid and stearidonic acid were epoxidised using m-chloroperbenzoic acid to achieve nine cis-monoepoxy-C18 fatty acid methyl esters (FAMEs), including novel methyl cis-monoepoxy derivatives of stearidonic acid and a cis-6,7-epoxy derivative of gamma-linolenic acid. These nine monoepoxy FAMEs were purified by normal-phase HPLC, identified by LC-MS, 1H and 13C NMR, and characterized by mass spectrometry and NMR spectroscopy. This study is focused on structural characterization of these C18 monoepoxy FAMEs using techniques in NMR spectroscopy including 1H, 13C, 1H-1H correlated spectroscopy (COSY) and 1H-13C heteronuclear correlation (HETCOR). The proton and carbon NMR chemical shifts of the epoxide, the double bonds, and the interrupted methylenes are assigned. Also discussed is an interpretation of the 1H and 13C NMR spectra of these monoepoxides including the changes in the 13C resonance of the olefinic carbons on the neighboring double bonds resulting from epoxide formation.     

Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03

A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced. In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found. Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile. The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis. The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain. Addition of glycerol to a solution containing Lip8 stabilized this enzyme. By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids.     

Methionine methyl group metabolism in lemna

To provide information upon the ways in which Lemna paucicostata uses the methyl group of methionine, plants were grown for various periods (from 1 minute to 6.8 days) in the presence of a tracer dose of radioactive methyl-labeled methionine. Protein methionine accounted for approximately 19% of the accumulated methyl moieties; other methylated products, about 81%. The latter group included (percent of total methyl in parentheses): methylated ethanolamine derivatives (46%); methyl esters of the pellet (chiefly, or solely, pectin methyl esters) (15%); chlorophyll methyl esters (8%); unidentified neutral lipids (6%); nucleic acid derivatives (2-5%); methylated basic amino acids (2%). No other major methylated compounds were observed in any plant fraction. Available evidence suggests that little, if any, oxidation of the methyl group of methionine, directly or indirectly, occurs in Lemna. Our results indicate that S-methyl-methionine sulfonium is formed relatively rapidly, but does not accumulate at a commensurate rate, probably being reconverted to methionine. To our knowledge, this is the first time a reasonably complete accounting of the metabolic fate of methionine methyl has been obtained for any plant. The extent to which the results with Lemna may be representative of the situation for other higher plants is discussed.     

Steric analysis of 3-, ω4-, ω3- and ω2-hydroxy acids and various alkanols by gas-liquid chromatography

D-Phenylpropionate (PP) derivatives of racemic hydroxy acid methyl esters and alkanols were prepared and the gas-liquid chromatographic separation of diastereoisomers was investigated using QF-1 as stationary phase. Good separations were obtained for the diasteroisomeric PP-derivatives of methyl 3-, 15-, 16-, and 17-hydroxyoctadecanoates. Less separation was observed for methyl 2- and 14-hydroxyoctadecanoates as PP-derivatives and there was no visible separation for PP-derivatives of 4-, 7-, or 13-hydroxyoctadecanoic acid methyl esters. The use of optically active 15-, 16- and 17-hydroxyoctadecanoic acids showed, that in these cases, the diastereoisomers containing L-hydroxy acids had shorter retention times than the ones containing D-hydroxy acids. On the other hand, the D-phenylpropionate derivative of methyl 3D-hydroxydecanoate had shorter retention time than the derivative of its L-enantiomer. PP-derivatives of 3-hexanol, 3-heptanol, 3-octanol, 2-octanol and 2-eicosanol could be resolved by gas-liquid chromatography.     

Beckmann rearrangement products of methyl 3-oxo-tirucall-8, 24-dien-21-oate from Boswellia serrata gum and their anti-tumor activity

3-Oxo-tirucall-8, 24-dien-3-one-21-oic acid is a minor natural product isolated from Boswellia serrata gum apart from β-boswellic acids. Since oxidation of 3-hydroxy group of β-boswellic acids leads to unstable beta-keto acids, Beckmann rearrangement could not be tried. Hence A-ring modified 3-oxo-tirucall-8, 24-dien-21 methyl esters (2–6) were synthesized for the first time via Beckmann rearrangement and evaluated for their anticancer potential against five human cancer cell lines (MCF-7, SW-982, HeLa, PC-3 and IMR-132) by MTT assay. While naturally occurring 3-oxo-tirucallic acid (1) and its methyl ester (2) exhibited nearly the same antiproliferative activity, A-ring modified molecules displayed improved anti-tumor activity with methyl A-homo-4-aza-3-oxo-tirucall-8, 24, dien-3-one-21-oate (4) exhibiting significant effect against prostate cancer cell lines.     

Design, synthesis, and antitumor evaluation of novel acenaphtho[1,2-b]pyrrole-carboxylic acid esters with amino chain substitution

8-Oxo-8H-acenaphtho[1,2-b]pyrrole-9-carboxylic acid esters and derivatives were prepared and evaluated for cytotoxicity against A549 and P388 cell lines. Based on a novel chromophore precursor 8-oxo-8H-acenaphtho[1,2-b]pyrrol-9-carbonitrile 1, the very insoluble 1 was converted to more soluble esters 5 and a series of 3-amino derivatives from 5 were obtained by mild S(N)Ar(H) reaction between 5 and various amines. The biological evaluation indicated that methyl esters 5a are the most cytotoxic with IC(50) values of 0.45 and 0.80 microM (against A549 and P388, respectively) among the parent esters 5a-5f, but 3-amino derivatives 4b and 4c of 5f with bromine showed the highest activity (with IC(50) values of 0.019-0.60 microM) among the 3-amino derivatives.