Properties of hopanoids and phosphatidylcholines containing ω-cyclohexane fatty acid in monolayer and liposome experiments

1,2,3,4-Tetrahydroxypentane-29-hopane (THBH) and a glycolipid derived from it are associated with ω-cyclohexane fatty acids-containing lipids in the membrane of Bacillus acidocaldarius. In order to elucidate the function of these lipids we studied mixed monolayer films and compared these with cholesterol-containing films. The hopanoids are able to condense a liquid-expanded film of di-ω-cyclohexyldodecanoylphosphatidylcholine (DCDPC). The condensing effect of THBH is smaller than that of cholesterol. Hopane glycolipid in comparison shows only little condensation. In a more condensed film, at lower temperatures, THBH slightly decreases while hopane glycolipid increases the molecular area. In egg phosphatidylcholine liposomes, 22-hydroxyhopane (diplopterol) and hopane glycolipid reduce the glycerol permeability to a smaller extent than cholesterol. In DCDPC liposomes, the effect of 22-hydroxyhopane is similar to that of cholesterol, while the hopane glycolipid shows only a weak reduction of the permeability. The results demonstrate that hepanoids have a cholesterol-like function in membranes. This function is also discussed in the context of membrane adaptation of a thermoacidophilic bacterium.     

Properties of Bacillus acidocaldarius mutants deficient in ω-cyclohexyl fatty acid biosynthesis

Mutants of the thermoacidophilic Bacillus acidocaldarius, auxotrophic for shikimate or cyclohyxyl-carboxylate, were isolated and characterized. The cyclohexylcarboxylate auxotrophs could be divided by crossfeeding experiments into two groups according to their genetic block. The cyclohexylcarboxylate auxotrophs were deficient in -cyclohexyl fatty acid biosynthesis. If the mutants were fed with branched-chain amino acids or short branched-chain fatty acids instead of cyclohexylcarboxylate they form a fatty acid pattern consisting of branched-chain fatty acids. In the high temperature/low pH range the growth yield of cells with this fatty acid pattern is lower as compared to wild type cells or mutants fed with cyclohexylcarboxylate. The same cells are also more sensitive to heat shocks and ethanol. The transport systems for lysine, glutamate and glucose are severely altered by the fatty acid pattern. It was also shown that the density of the lipids containing -cyclohexyl fatty acids is higher compared to cells with branched-chain fatty acids. Thus it could be supposed that this alteration influences transport systmes in a direct manner or via energization of the cytoplasmic membrane.     

Occurrence of Curtobacterium sp. possessing ω-cyclohexyl fatty acids in soil with zinc added

A bacterial strain, Curtobacterium sp., isolated from a soil with zinc added possessed -cyclohexyl fatty acids. -Cyclohexyl undecanoic acid made up 47% of the total fatty acids; it was the most abundant fatty acid in the strain grown in tryptone medium. 12-Methyl tetradecanoic acid (23%) and 14-methyl hexadecanoic acid (22%) were also major fatty acids. The proportion of -cyclohexyl undecanoic acid increased as the pH of the medium decreased and as the culture temperature increased.The bacteria grew almost normally in zinc-enriched medium, and -cyclohexyl undecanoic acid increased with zinc concentration. Zinc added to the medium was not abundant in the cell fraction, and the ratio of increase of zinc in the cells was not so high as in the culture medium. These results suggested that -cyclohexyl fatty acids are related to the zinc tolerance of the isolated strain, and that this tolerance depends on low permeability of the membrane to zinc.     

Synthesis,electrochemical and spectral properties of some ω-N-quinonyl amino acids

Summary. Four series of ω-N-quinonyl amino acids were synthesized by Michael-like additions. The quinones include 2-phenylthio-1,4-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone and 2,3-dichloro-1,4-naphthoquinone. These modified amino acids can be used for post chain assembly modifications of biologically active peptides, which target the quinonic drug to a cancer damaged area. The electron-transfer capabilities of the modified amino acids were probed by cyclic voltammetry measurements. The results described in this paper show that the novel N-quinonyl amino acids are effective in producing semiquinone radicals similarly to the unconjugated quinones themselves. A direct relation was found between the first reduction potentials of the quinones and their reactivity towards the ω-amino acids. The successful generation of stable semiquinone radicals by the novel quinone derivatives is a prerequisite for the manifestation of site-directed antitumor activity of corresponding quinone-peptide conjugates. Received January 3, 2001 Accepted March 28, 2001     

The genus Dracunculus--a source of triacylglycerols containing odd-numbered ω-phenyl fatty acids

Reversed phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/MS-APCI) was used to identify and quantify triacylglycerols (TAGs) having odd-numbered ω-phenylalkanoic acids from seeds of the flower plant Dracunculus vulgaris, and TAGs from the bacterium Rhodococcus erythropolis prepared by precursor directed biosynthesis from phenylalanine and having the corresponding even-numbered ω-phenylalkanoic acids. Model compounds, which are not commercially available, were prepared by organic synthesis and this allowed us to extend the number of identified natural TAGs to nearly 140 molecular species. Both synthetic and natural compounds containing ω-phenylalkanoic acids were found to have antioxidant and free radical scavenging properties.     

Synthesis and binding properties of endomorphin-2analogs containing α-hydroxymethyl amino acids

Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)--hydroxymethyltyrosine in position 1 and (R)- and (S)--hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that -hydroxymethyl substitution of aminoacid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.     

Effects of phospholipid acyl chain structure on thermotropic phase properties. 3. Phosphatidylcholines with (−), and (±)-anteiso acyl chains

We have synthesized a number of anteiso-branched fatty acids, both as racemates and as pure (?)-stereoisomers, as well as the corresponding di-anteiso-acyl phosphatidylcholines (PCs). The phase transition temperatures (_c of the hydrated PCs have been determined by differential thermal analysis (DTA). Consideration of the observed effects of acyl chain length and terminal see-butyl group configuration on anteiso acid melting points and di-anteiso-acyl PC transition temperatures leads us to the conclusion that the terminal branched portions of anteiso acyl chains interact only weakly with adjacent acyl chains in the hydrated phosphatidylcholine bilayer and probably in the anhydrous fatty acid crystal as well. In agreement with this conclusion, we observe that the DTA heating transition endotherms for hydrated di-anteiso-acyl PCs appear to be considerably less strongly endothermic, and occur at much lower temperatures, than do the major transition endotherms for the di-n-acyl PCs of like carbon number. Our findings generally support the proposal that anteiso acyl lipids tend to fluidize membranes, although they do so less effectively than do cis-unsaturated acyl lipids.     

Synthesis and bioevaluation of ω-N-amino analogs of B13

Novel ω-N-amino analogs of B13 (Class E) were designed, synthesized and tested as inhibitors of acid ceramidase (ACDase) and potential anticancer agents deprived of unwanted lysosomal destabilization and ACDase proteolytic degradation properties of LCL204 [Szulc, Z. M.; Mayroo, N.; Bai, A.; Bielawski, J.; Liu, X.; Norris, J. S.; Hannun, Y. A.; Bielawska, A. Bioorg. Med. Chem. 2008, 16, 1015].Representative analog LCL464, (1R,2R)-2-N-(12′-N,N-dimethylaminododecanoyl amino)-1-(4″-nitrophenyl)-1,3-propandiol, inhibited ACDase activity in vitro, with a similar potency as B13 but higher than LCL204. LCL464 caused an early inhibition of this enzyme at a cellular level corresponding to decrease of sphingosine and specific increase of C₁₄- and C₁₆-ceramide. LCL464 did not induce lysosomal destabilization nor degradation of ACDase, showed increased cell death demonstrating inherent anticancer activity in a wide range of different cancer cell lines, and induction of apoptosis via executioner caspases activation. LCL464 represents a novel structural lead as chemotherapeutic agent acting via the inhibition of ACDase.     

Purification and physical properties of sweet-almond α-galactosidase

1. α-Galactosidase from sweet almonds was purified about 2000-fold through eight steps. 2. The enzyme preparation was free from other related enzymes known to occur in sweet almonds, and behaved as a homogeneous protein on filtration through Sephadex G-75. 3. A molecular weight of about 33000 was determined from the gel-filtration data. 4. The ultraviolet-absorption spectrum and thermal inactivation of the enzyme are described. 5. The purified enzyme hydrolysed p-nitrophenyl α-d-galactoside at a much faster rate than melibiose. 6. The pH optimum was at 5·5–5·7. 7. Besides hydrolysis, it also catalysed transfer of galactosyl residues, chain elongation of melibiose and the synthesis of oligosaccharides from galactose.     

Chemical synthesis and biological evaluation of ω-hydroxy polyunsaturated fatty acids

ω-Hydroxy polyunsaturated fatty acids (PUFAs), natural metabolites from arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were prepared via convergent synthesis approach using two key steps: Cu-mediated CC bond formation to construct methylene skipped poly-ynes and a partial alkyne hydrogenation where the presence of excess 2-methyl-2-butene as an additive that is proven to be critical for the success of partial reduction of the poly-ynes to the corresponding cis-alkenes without over-hydrogenation. The potential biological function of ω-hydroxy PUFAs in pain was evaluated in naive rats. Following intraplantar injection, 20-hydroxyeicosatetraenoic acid (20-HETE, ω-hydroxy ARA) generated an acute decrease in paw withdrawal thresholds in a mechanical nociceptive assay indicating pain, but no change was observed from rats which received either 20-hydroxyeicosapentaenoic acid (20-HEPE, ω-hydroxy EPA) or 22-hydroxydocosahexaenoic acid (22-HDoHE, ω-hydroxy DHA). We also found that both 20-HEPE and 22-HDoHE are more potent than 20-HETE to activate murine transient receptor potential vanilloid receptor1 (mTRPV1).     

2′-modified oligoribonucleotides containing 1,2-diol and aldehyde groups. Synthesis and properties

1,2-Diol-oligoribonucleotides were prepared using fully protected 2??-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine 3??-phosphoramidite. Incorporation of the modified uridine residue into oligonucleotide chains was not shown to significantly affect the thermal stability of RNA-RNA and RNA-DNA duplexes. Periodate oxidation of the 1,2-diol group resulted in reactive 2??-aldehyde oligoribonucleotides. These oligonucleotides were studied for their application in the affinity modification of RNA recognizing proteins with an example of bacterial ribonuclease P.     

Oxidation of ω-hydroxylated fatty acids and steroids by SS-isoenzyme of liver alcohol dehydrogenase

The SS-isoenzyme of alcohol dehydrogenase from horse liver was found to be active towards ω1- and ω2-hydroxylated fatty acids, an ω-hydroxylated steroid, ethanol and a 3β-hydroxysteroid. The main part of all these activities disappeared after carboxymethylation of a cysteineresidue at the active site of LADH_SS. The ω-hydroxyfatty acid dehydrogenase activity of LADH_SS was of similar magnitude as that of LADH_EE whereas the ω-hydroxysteroid dehydrogenase activity of LADH_SS was considerably higher than that of LADH_EE.     

Identification of free radicals of glycerophosphatidylcholines containing ω-6 fatty acids using spin trapping coupled with tandem mass spectrometry

Metal-catalysed radical oxidation of diacyl-glycerophosphatidylcholines (GPC) with ω-6 acyl polyunsaturated fatty acids (PAPC, palmitoyl-arachidonoyl-glycerophosphatidylcholine and PLPC, palmitoyl-lineloyl-glycerophosphatidylcholine) was studied. Free radical oxidation products were trapped by spin trapping with 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) and identified by electrospray mass spectrometry (ES-MS). The spin adducts of oxidised GPC containing one and two oxygen atoms and one and two DMPO molecules were observed as doubly charged ions. Structural characterisation by tandem mass spectrometry (MS/MS) of these ions revealed product ions corresponding to loss of the acyl chains (sn-1-palmitoyl and sn-2-oxidised spin adduct of lineloyl or arachidonoyl), loss of the spin trap (DMPO) and product ions attributed to oxidised sn-2 fatty acid spin adduct (lineloyl and arachidonoyl). Product ions formed by homolytic cleavages near the spin trap and also from 1,4 hydrogen elimination cleavages involving the hydroxy group in the sn-2 fatty acid spin adduct allowed to infer the nature of the radical. Altogether, the presence of GPC hydroxy-alkyl/DMPO and hydroxy-alkoxyl/DMPO spin adducts was proposed.     

Higher de novo synthesized fatty acids and lower ω3- and ω6-long-chain polyunsaturated fatty acids in umbilical vessels of women with preeclampsia and high fish intakes

Umbilical veins (UV) and arteries (UA) of preeclamptic women in Curaçao harbor lower long-chain polyunsaturated fatty acids (LCP). The present aim was to test these findings in Mwanza (Tanzania), whose inhabitants have high LCPω3 and LCPω6 intakes from Lake Victoria fish. Women with preeclampsia (n=28) in Mwanza had lower PUFA and higher 20:0 in UV and UA, compared with normotensive/non-proteinuric controls (n=31). Their UV 22:6ω3, 22:4ω6, LCPω6, ω6, and LCPω3+ω6 were lower, while saturated FA, potentially de novo synthesized FA (Σde novo) and (Σde novo)/(LCPω3+ω6) ratio were higher. Their UA had higher 16:1ω7, ω7, 18:0, and 16:1ω7/16:0. Umbilical vessels in Mwanza had higher 22:6ω3, LCPω3, ω3, and 16:0, and lower 22:5ω6, 20:2ω6, 18:1ω9, and ω9, compared to those in Curaçao. Preeclampsia in both Mwanza and Curaçao is characterized by lower LCP and higher Σde novo. An explanation of this might be placental dysfunction, while the similarity of umbilical vessel FA-abnormalities in preeclamptic and diabetic pregnancies suggests insulin resistance as a common denominator.     

Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid composed of 22 carbon atoms and six double bonds. Because the first double bond, as counted from the methyl terminus, is at position three, DHA belongs to the so-called -3 group. In recent years, DHA has attracted much attention because of its beneficial effect on human health. At present, fish oil is the major source of DHA, but alternatively it may be produced by use of microorganisms. Marine microorganisms may contain large quantities of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light. These processes can be well controlled and DHA with constant quality can be produced all year round. This paper reviews recent advances in the biotechnological production of DHA by marine microorganisms.     

Synthesis, crystal structures and spectroscopic properties of copper(I) complexes containing β-dialdiminato supporting ligands

Two mononuclear neutral copper(I) complexes, Cu(L¹)PPh₃ (1), Cu(L²)(PPh₃)₂ (2) ([L¹]⁻ = [{N(C₆H₃ⁱPr₂-2,6)C(H)}₂CPh]⁻; [L²]⁻ = [{N(C₆H₅)C(H)}₂CPh]⁻) have been synthesized and structurally characterized by X-ray crystallography. In complex 1, the copper(I) atom is in a distorted three-coordinate trigonal planar environment, whereas in complex 2 with the less sterically hindered β-dialdiminato ligand, the copper(I) atom is the centre of a four-coordinate distorted tetrahedron. At room temperature complexes 1 and 2 in a film of PMMA exhibit green emission at 543 and 549 nm with lifetimes of 5.28 and 5.32 ns, respectively.     

Toxic fluorine compounds. The use of the ω-fluorine atom in the study of the metabolism of fatty acids containing ethyl, n-propyl and isopropyl branches

1. Some omega-fluoroalkanoic acids containing a branched ethyl, n-propyl or isopropyl group in the alpha- or beta-position have been prepared. 2. A study of the toxicity of these acids revealed that the ethyl group partially inhibited, and the n-propyl and isopropyl groups completely inhibited, the normal processes of oxidative degradation.     

Regulating effect of β-ketoacyl synthase domain of fatty acid synthase on fatty acyl chain length in de novo fatty acid synthesis

Fatty acid synthase (FAS) is a multifunctional homodimeric protein, and is the key enzyme required for the anabolic conversion of dietary carbohydrates to fatty acids. FAS synthesizes long-chain fatty acids from three substrates: acetyl-CoA as a primer, malonyl-CoA as a 2 carbon donor, and NADPH for reduction. The entire reaction is composed of numerous sequential steps, each catalyzed by a specific functional domain of the enzyme. FAS comprises seven different functional domains, among which the β-ketoacyl synthase (KS) domain carries out the key condensation reaction to elongate the length of fatty acid chain. Acyl tail length controlled fatty acid synthesis in eukaryotes is a classic example of how a chain building multienzyme works. Different hypotheses have been put forward to explain how those sub-units of FAS are orchestrated to produce fatty acids with proper molecular weight. In the present study, molecular dynamic simulation based binding free energy calculation and access tunnels analysis showed that the C16 acyl tail fatty acid, the major product of FAS, fits to the active site on KS domain better than any other substrates. These simulations supported a new hypothesis about the mechanism of fatty acid production ratio: the geometric shape of active site on KS domain might play a determinate role.     

Synthesis and antidiabetic performance of β-amino ketone containing nabumetone moiety

We wish to report the further design and improved synthesis that resulted in two series of target molecules, TM-1 and TM-2, with remarkably simplified structures containing β-amino ketone of discrete nabumetone moiety. These were obtained via a 'one-pot, two-step, three-component' protocol of Mannich reaction with yield up to 97%. A total of 28 out of 31 new compounds were characterized using (1)H NMR, (13)C NMR, ESI MS and HRMS techniques. Studies on their antidiabetic activities, screened in vitro at 10 μg mL(-1) level, indicate that TM-2 possesses peroxisome proliferator-activated receptor activation and α-glucosidase inhibition activity significantly stronger than that of TM-1, and also that of the series B compounds that were previously synthesized by the group. Analysis of the structure-activity relationship points to the sulfanilamide unit as the most probable potent group of β-amino ketone and, on the basis of which, a tangible strategy is presented for the development of new antidiabetic drugs.     

Activity and stability of different immobilized preparations of recombinant E. coli cells containing ω-transaminase

Production of chiral amines using ω-transaminases has been thoroughly studied in recent years. Immobilized ω-transaminases, however, have been used on relatively few occasions despite potential benefits such as reuse of enzyme and ease of product purification. In this study principally different methods including surface immobilization, entrapment and sweep flocculation using titanium oxide, Ca-alginate and chitosan respectively were evaluated for the immobilization of recombinant Escherichia coli cells. The enzyme expressed was a modified Arthrobacter citreus ω-transaminase with improved thermostability. The preparations were compared in terms of cell loading capacity, operational stability in repeated batches and storage stability using the conversion of methylbenzylamine to acetophenone.The use of chitosan for cell immobilization proved to be the method of choice since it was both very simple and effective. At a very high cell loading of 3.2 g cells/g chitosan >60% activity was observed. The preparation was reused in eight successive 1-h batches with >90% remaining activity. To further demonstrate its usability the preparation was used for asymmetric synthesis of (S)-4′-cyano-(α)-methylbenzylamine in three repeated bathes (cycle time >20 h), using isopropylamine as the amine donor. Storage stability was comparable with that of non-immobilized cells. It was concluded that the chitosan method due to its properties and simplicity would be advantageous for use also on a larger scale.