Computer study of intramolecular ordering in octadecatriene chains with cis-double bonds

Computer simulations of isolated unperturbed hydrocarbon molecules of C18:3 with methylene-interrupted cis double bonds were carried out using the Monte Carlo method based on the continuum model. A molecule-fixed coordinate system (with the axes along the principal axes of inertia of each molecule conformation) was used. The orientation distribution functions rho and order parameters S for C-H and C-C bonds relative to the maximum molecule span axis were calculated. It was shown that the widths of functions rho (factor of bond "fluctuations") are dependent on the chemical structure and position of the segment, fluctuations increase from the centre of the chain towards the terminals, all things being equal. The orientation distributions rho of C-H bonds flanking the double bond are the most narrow, the functions rho of CH2-groups located between two double bonds are the widest. It turned out that order parameter -SCH profiles of isolated chains of C18:3 include both the positive and negative values. The parameter SCC odd-even effect in unsaturated molecules of such structure changes the sign between double bonds.     

Polyunsaturated hydrocarbon chains: computer study of the characteristics of intramolecular ordering of chains

The conformational properties of isolated unbranched hydrocarbon polyunsaturated molecules of cis-C18:4 and cis-C18:5 under theta-conditions (T = 298 K) were studied using Monte Carlo simulations. The conformations were generated by a computer (the continuum model was used; the energy of nonbonded interactions and torsion and electrostatic terms were taken into account). A molecule-fixed coordinate system with the axes along inertia tensor eigenvectors of each molecule conformation (principal axes of inertia) were used for the calculations. C-H and C-C bond orientation distribution functions rho and ordering parameters S with respect to the maximum molecule span axis were calculated. It was shown that the presence of five methylene-interrupted cis double bonds in C18 chain has a maximum effect on the intramolecular ordering properties of the molecule. The widths of function rho CH for pentaenes differed significantly from those of other C18-chains: the widths of function rho for all CH2-groups were nearly twice as large as that for C-H-bonds flanking the double bonds C=C, and roughly constant along the chain sequence. The mean magnitudes of magnitudes of SCH in the molecule decreased when unsaturation increased.     

Molecular Dynamics Investigation of Bond Ordering of Unsaturated Lipids in Monolayers

Molecular dynamics simulations of three model lipid monolayers of 2,3-diacyl-D-glycerolipids, that contained stearoyl (18:0) in the position 3 and oleoyl (18:9cis), linoleoyl (18:26cis), or linolenoyl (18:33cis) in the position 2, have been carried out. The simulation systems consisted of 24 lipid molecules arranged in a rectangular simulation cell, with periodic boundary conditions in the surface plane. 1 nanosecond simulations were performed at T = 295 K. C-C and C-H bond order parameter profiles and the bond orientation distributions about the monolayer normal have been calculated. The relation of the distributions to the order parameters was analyzed in terms of maxima and widths of the distributions. The cis double bond order parameter is found to be higher than those of adjacent single C-C bonds. The widths of the two distributions of C-H bonds of the cis double bond segment in di- and triunsaturated molecules are much smaller than that obtained for methylene group located between the double bonds. The bond orientation distribution function widths depend on both the segment location in the chain and the segment chemical structure.     

Effect of cholesterol on the bond ordering in hydrated unsaturated lipid bilayers

Molecular dynamics computer simulations of hydrated bilayers of unsaturated phosphatidylcholines in which double bonds are in the states: 18:0/18:1(n-9)cis (PC), 18:0/18:2(n-6)cis (PC), 18:0/18:3(n-3)cis (PC), 18:0/20:4(n-6)cis (PC), and 18:0/22:6(n-3)cis in the presence of cholesterol (40 mol%) and its absence have been performed. The simulation have been performed at 303 K and 1 atm, under the conditions corresponding to the experimentally observed liquid-crystalline state of the bilayer from phosphatidylcholine. The C-C and C-H bond order parameter profiles with respect to the bilayer normal and the C-C bond orientation distribution functions have been calculated. The widths of the functions and positions of their maxima have been determined. The dependence of these characteristics on the type of the bond, the degree of unsaturation of the chain, the presence of cholesterol in the bilayer, and the bond order parameters have been analyzed.     

Specificities of the Acyl-Acyl Carrier Protein (ACP) Thioesterase and Glycerol-3-Phosphate Acyltransferase for Octadecenoyl-ACP Isomers (Identification of a Petroselinoyl-ACP Thioesterase in Umbelliferae)

This study was designed to address the question: How specific for double bond position and conformation are plant enzymes that act on oleoyl-acyl carrier protein (ACP)? Octadecenoyl-ACPs with cis double bonds at positions [delta]6, [delta]7, [delta]8, [delta]9, [delta]10, [delta]11, or [delta]12 and elaidyl (18:1[delta]9trans)-ACP were synthesized and used to characterize the substrate specificity of the acyl-ACP thioesterase and acyl-ACP:sn-glycerol-3-phosphate acyltransferase. The two enzymes were found to be specific for the [delta]9 position of the double bond. The thioesterase was highly specific for the [delta]9 cis conformation, but the transferase was almost equally active with the cis and the trans isomer of 18:1[delta]9-ACP. In plants such as the Umbelliferae species coriander (Coriandrum sativum L.) that accumulate petroselinic acid (18:1[delta]6cis) in their seed triacylglycerols, a high petroselinoyl-ACP thioesterase activity was found in addition to the oleoyl-ACP thioesterase. The two activities could be separated by anion-exchange chromatography, indicating that the petroselinoyl-ACP thioesterase is represented by a distinct polypeptide.     

Water permeability and mechanical strength of polyunsaturated lipid bilayers

Micropipette aspiration was used to test mechanical strength and water permeability of giant-fluid bilayer vesicles composed of polyunsaturated phosphatidylcholine PC lipids. Eight synthetic-diacyl PCs were chosen with 18 carbon chains and degrees of unsaturation that ranged from one double bond (C18:0/1, C18:1/0) to six double bonds per PC molecule (diC18:3). Produced by increasing pipette pressurization, membrane tensions for lysis of single vesicles at 21 degrees C ranged from approximately 9 to 10 mN/m for mono- and dimono-unsaturated PCs (18:0/1, 18:1/0, and diC18:1) but dropped abruptly to approximately 5 mN/m when one or both PC chains contained two cis-double bonds (C18:0/2 and diC18:2) and even lower approximately 3 mN/m for diC18:3. Driven by osmotic filtration following transfer of individual vesicles to a hypertonic environment, the apparent coefficient for water permeability at 21 degrees C varied modestly in a range from approximately 30 to 40 microm/s for mono- and dimono-unsaturated PCs. However, with two or more cis-double bonds in a chain, the apparent permeability rose to approximately 50 microm/s for C18:0/2, then strikingly to approximately 90 microm/s for diC18:2 and approximately 150 microm/s for diC18:3. The measurements of water permeability were found to scale exponentially with the reduced temperatures reported for these lipids in the literature. The correlation supports the concept that increase in free volume acquired in thermal expansion above the main gel-liquid crystal transition of a bilayer is a major factor in water transport. Taken together, the prominent changes in lysis tension and water permeability indicate that major changes occur in chain packing and cohesive interactions when two or more cis-double bonds alternate with saturated bonds along a chain.     

19F nuclear magnetic resonance studies of lipid fatty acyl chain order and dynamics in Acholeplasma laidlawii B membranes. Orientational order in the presence of a series of positional isomers of cis-octadecenoic acid

The 19F nuclear magnetic resonance (NMR) spectra of membranes of Acholeplasma laidlawii B enriched with one of a series of positional isomers of cis-octadecenoic acid plus small amounts of one of a number of isomers of monofluoropalmitic acid were interpreted in terms of an orientational order parameter (Smol). The variation of Smol with the position of the fluorine label in the liquid-crystalline state yielded an "order profile" with characteristics similar to those obtained via 2H NMR and which was relatively invariant regardless of the site of cis unsaturation. In the gel state, values of Smol approached the theoretical maximum, and the order profiles in the presence of different isomeric cis-octadecenoic acids displayed distinct dissimilarities. When the cis double bond was located proximal to the methyl terminus of the fatty acyl chain, a gradient of order across the bilayer was still evident in the gel state. When the cis double bond was located near the carbonyl head group, values of Smol were approximately equal at all chain positions. These observations were interpreted as indicating that in the gel state the stringency of packing restrictions is still subject to variation across the width of the bilayer. Relative overall orientational order among all isomers examined (specifically, 18:1c delta 4, delta 5, delta 6, delta 7, delta 8, delta 9, delta 10, delta 11, delta 12, delta 13, delta 14, and delta 15) varied directly as a function of proximity to the lipid gel to liquid-crystalline phase transition (Tm) (determined via differential scanning calorimetry) when compared at a constant temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Raman studies of some diunsaturated olefinic and acetylenic fatty acids and their derivatives.

The Raman spectra of CCl4 solutions of the 6,12; 7,12; 8,12; 9,12; and 10,12 isomers of octadecadiynoic acid and of the octadecadienoic acid methyl esters of both the cis,cis and trans,trans series are reported. Provided that there are two or more methylene groups between the unsaturated groups, the double and triple bond vibrational wavenumber values are close to those found in monounsaturated derivatives. An attempt has been made to obtain a correlation between the relative intensities of the nu(CequalsC) and delta(CH2) bands and the ratio of the number of double bonds to methylene groups in the molecule.     

Unsaturated fatty acid isomers: effects on the circadian rhythm of a fatty-acid-deficient Neurospora crassa mutant

The fatty acids oleic, linoleic, and linolenic, each of which has a cis double bond at the delta 9 position, are known to lengthen the circadian period of conidiation (spore formation) of strains of Neurospora crassa carrying the cel mutation. cel confers a partial fatty acid requirement on the organism and has been used to promote incorporation of exogenous fatty acids. To test whether a physical effect imparted by the cis double bonds, such as increased membrane fluidity, is critical for the perturbation of the rhythm, various isomers of these fatty acids were supplemented to the bd csp cel strain. Positional isomers of oleic acid, such as petroselinic (delta 6) and vaccenic (delta 11) acids, and longer-chain isomers, such as eicosenoic (delta 11) and erucic (delta 13) acids, did not lengthen the rhythm. The shorter-chain palmitoleic (delta 9) acid did not give a consistent lengthening of the rhythm; it may be elongated to vaccenic acid. In contrast, gamma-linolenic acid (delta 6,9,12) dramatically lengthened the period. Linoelaidic acid (the trans,trans isomer of linoleic acid) lengthened the period at 22 degrees C, but elaidic acid (the trans isomer of oleic acid) did not. Elaidic acid was shown to exert a lengthening effect, but only at lower temperatures. The data do not support a direct physical action as the source of the fatty acids' "chronobiotic" ability.     

Backbone modifications in cyclic peptides. Conformational analysis of a cyclic pseudopentapeptide containing a thiomethylene ether amide bond replacement

NMR and X-ray crystallographic studies have shown that cyclic pentapeptides of the general structure cyclo(D-Xxx-Pro-Gly-Pro-Gly) possess beta- and gamma-turn intramolecular hydrogen bonds. As part of our continuing series surveying the compatibility of various amide bond replacements on peptide structure, we have synthesized cyclo(D-Phe-Pro psi[CH2S]Gly-Pro-Gly). The pseudopeptide was prepared by solid phase methods and cleaved from the resin by a new procedure involving phase transfer catalysis using K2CO3 and tetrabutylammonium hydrogen sulfate. Cyclization was carried out with the use of DPPA, HOBt, and DMAP to afford the product in 69% yield. The conformational behavior of the pseudopeptide was analyzed by 1H and 13C (1D and 2D) NMR techniques. The backbone modification replaced the amide bond that is involved in a gamma-turn intramolecular hydrogen bond in the all-amide structure. In CDCl3, the pseudopeptide adopted the same all-trans conformation as its parent, although the remaining beta-turn hydrogen bond was weaker according to delta delta/delta TNH measurements. In DMSO-d6, the all-trans conformer and a second conformer were observed in a ratio of 55:45. These conformers, which slowly interconverted on the NMR time scale, could be separately assigned; peaks due to chemical exchange were readily distinguishable by the ROESY technique as reported earlier by others. 13C and ROESY experiments suggested the minor conformer contained one cis amide bond at the Gly1-Pro2 position. Thus, both the location and type of amide surrogate are important determinants affecting the compatibility of the replacement with a particular conformational feature.     

Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid

Divergent forms of the plant Delta(12)-oleic-acid desaturase (FAD2) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta(11),Delta(13)-double bonds by modification of an existing Delta(12)-double bond in C(18) fatty acids. Here, we report a class of FAD2-related enzymes that modifies a Delta(9)-double bond to produce the conjugated trans-Delta(8),trans-Delta(10)-double bonds found in calendic acid (18:3Delta(8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis. Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C. officinalis developing seed cDNA library. The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-2 in somatic soybean embryos resulted in the production of calendic acid. In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) of the total fatty acids. In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18:2Delta(9cis,12cis)). These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta(9)-double bond of linoleic acid. Regiospecificity for Delta(9)-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.     

Quantitative effects of unsaturated fatty acids in microbial mutants. VII. Influence of the acetylenic bond location on the effectiveness of acyl chains.

The ability of a series of 18 carbon acetylenic fatty acids to fulfill the unsaturated fatty acid requirements of Escherichia coli and Saccharomyces cerevisiae was investigated. Despite their high melting points (greater than 40 degrees C), several isomers of the acetylenic fatty acids were as efficient or more efficient in supporting growth than the analogous fatty acid having a cis-double bond. The efficiencies of the different positional isomers in supporting cell proliferation varied from essentially 0 cells per fmol for the 2-5 and 13-17 isomers to high values when the acetylenic bond was near the center of the chain: e.g. 45 E. coli and 5.5 S. cerevisiae cells/fmol for the 10 isomer. A striking ineffectiveness of the 9 isomer was observed with E. coli. The 7, 8 and 10 isomers were at least 10-fold more efficient than any of the other positional isomers in supporting the growth of E. coli. In contrast, the 9 isomer was among the most effective acetylenic fatty acids tested with the yeast mutant. Chromatographic analysis of the extracted lipids indicated that each of the acetylenic isomers tested (except delta2 and delta3) could be esterified by the prokaryotic and eukaryotic microorganisms. The content of unsaturated plus cyclopropane acids observed when growth ceased in E. coli cultures supplemented with growth-limiting concentrations of the acetylenic fatty acids ranged from approx. 15 mol% for the 8 isomer to approx. 35 mol% for the 14 and 17 isomers. The 8-11 isomers were observed to be esterified predominantly at the two position in phosphatidylethanolamine of E. coli and in phosphatidylcholine of S. cerevisiae.     

An unsaturated fatty acid mutant of Aspergillus niger with partially defective delta 9-desaturase

The wild-type Aspergillus niger (V35) does not require fatty acids for growth. Four unsaturated fatty acid auxotrophs designated as UFA1, UFA2, UFA3, and UFA4 have been produced from this organism by treating the conidia of the wild-type strain with a mutagen, N-methyl-N'-nitro-N-nitrosoguanidine, followed by isolation on media containing monounsaturated fatty acids and the nonionic detergent, Brij 58. Optimal growth of the mutants comparable with that of the wild type was achieved with medium supplemented with C16 or C18 unsaturated fatty acids containing at least one cis double bond at the delta 9 position. Some other fatty acids (18:1 delta 11 cis and 16:1 delta 9 trans) support growth to some extent. The mutants do not grow at all in the presence of saturated fatty acids. Fatty acid analyses of the mutant, UFA2, grown in the presence of different fatty acid supplements reveal that it may be defective in a desaturase system. Experiments with unlabeled and [1-14C]palmitoyl-CoA have shown that the microsomes of the mutant (UFA2) contain a partially defective delta 9-desaturase system.     

Fatty acid structural requirements for activity of arachidonoyl-CoA synthetase

We have examined the fatty acid substrate specificity of arachidonoyl-CoA synthetase from human platelet membranes. A variety of positional isomers and chain-length analogs of arachidonic acid [20:4(5, 8, 11, 14)] were synthesized, and assayed for their ability to inhibit arachidonoyl-CoA formation or to serve as substrates for the synthetase. The chain-length specificity of the synthetase for delta 8,11,14 trienoic fatty acids was C19 greater than C18 = C20 much greater than C21 greater C22. Inhibition activity by positional isomers of arachidonate was 20:4(5, 8, 11, 14) approximately equal to 20:4(6, 9, 12, 15) = 20:4(7, 10, 13, 16) much greater than 20:4(4, 7, 10, 13), however, Vmax for arachidonate was greater than that for 20:4(6, 9, 12, 15). The enzyme apparently "counts" double bonds from the carboxyl terminus. As counted from the methyl terminus we found that several n-6,-9,-12 fatty acids were ineffective as inhibitors [18:3(6, 9, 12); 19:4)4, 7, 10, 13); 21:3(9, 12, 15)], whereas all methylene-interrupted tri- and tetraenoic fatty acids which contained delta 8 and delta 11 double bonds were potent inhibitors. The delta 11 double bond was best associated with optimal inhibition: 20:3(5, 11, 14) had a lower Ki than 20:3(5, 8, 14). 13-Methyl-20:3(8, 11, 14) did not inhibit the enzyme. Partially purified enzyme from calf brain, depleted of nonspecific long-chain acyl-CoA synthetase, exhibited the same fatty acid specificity as crude platelet enzyme.     

Thermodynamics of hydrogen bond and hydrophobic interactions in cyclodextrin complexes.

Values of K, delta G(o), delta H(o), delta S(o) and delta C(po) for the binding reaction of small organic ligands forming 1:1 complexes with either alpha- or beta-cyclodextrin were obtained by titration calorimetry from 15 degrees C to 45 degrees C. A hydrogen bond or hydrophobic interaction was introduced by adding a single functional group to the ligand. The thermodynamics of binding with and without the added group are compared to estimate the contribution of the hydrogen bond or hydrophobic interaction. A change in the environment of a functional group is required to influence the binding thermodynamics, but molecular size-dependent solute-solvent interactions have no effect. For phenolic O-H-O hydrogen bond formation, delta H(o) varies from -2 to -1.4 kcal mol(-1) from 15 degrees C to 45 degrees C, and delta C(p) is increased by 18 cal K(-1) mol(-1). The hydrophobic interaction has an opposite effect: in alpha-cyclodextrin, delta C(po) = -13.3 cal K(-1) mol(-1) per ligand -CH(2)-, identical to values found for the transfer of a -CH(2)-group from water to a nonpolar environment. At room temperature, the hydrogen bond and the -CH(2)-interaction each contribute about -600 cal mol(-1) to the stability (delta G(o)) of the complex. With increased temperature, the hydrogen bond stability decreases (i.e., hydrogen bonds "melt"), but the stability of the hydrophobic interaction remains essentially constant.     

New antitumour cyclic astin analogues: synthesis, conformation and bioactivity.

Astins, antitumour cyclic pentapeptides, were isolated from the Aster tataricus. Their chemical structures, consist of a 16-membered ring system containing a unique beta,gamma-dichlorinated proline [Pro(Cl)2], other non-coded amino acid residues and a cis conformation in one of the peptide bonds. The astin backbone conformation, along with the cis peptide bond in which the beta,gamma-dichlorinated proline residue is involved, was considered to play an important role in their antineoplastic activities on sarcoma 180A and P388 lymphocytic leukaemia in mice, but the scope and potential applications of this activity remain unclear. With the aim at improving our knowledge of the conformational properties influencing the bioactivity in this class of compounds, new astin-related cyclopeptides were synthesized differing from the natural products by the presence of some non-proteinogenic amino acid residues: Aib, Abu, -(S)beta3-hPhe and a peptide bond surrogate (-SO2-NH-). The analogues prepared c(-Pro-Thr-Aib-beta3-Phe-Abu-), c[Pro-Thr-Aib-(S)beta3-hPhe-Abu], c[Pro-Abu-Ser-(S)beta3-hPhe psi(CH2-SO2-NH)-Abu] and c[Pro-Thr-Aib-(S)beta3-hPhe psi(CH2-SO2-NH)-Abu] were synthesized by classical methods in solution and tested for their antitumour effect. These molecules were studied by crystal-state x-ray diffraction analysis and/or solution NMR and MD techniques.     

Double-Bond Requirement in a Fatty Acid Desaturase Mutant of Saccharomyces cerevisiae

A fatty acid Delta(9)-desaturase mutant of yeast was analyzed to establish the specificity of the fatty acid structural requirement. Several double-bond-containing and substituted fatty acids were tested. It was concluded that the presence of a Delta(9)cis double bond is necessary for growth. The need for a specific chain length or a specific number of double bonds, or for both, is more flexible. Tracer-containing 16:1Delta(9)cis, 18:1Delta(9)cis, 18:2Delta(9,12)cis,cis and 18:3Delta(9,12,15) all cis revealed that each of these growth-supporting components, once taken up, was not converted into other fatty acids. Concentration effects on doubling time were also considered.     

Applications of natural orbitals for chemical valence in a description of bonding in conjugated molecules

Natural orbitals for chemical valence (NOCV) were used to describe bonding in conjugated pi-electron molecules. The 'single' C-C bond in trans-1,3-butadiene, 1,3-butadiene-1,1,4,4-tetra-carboxilic acid, 1,3,5,7-octatetraene, and 11-cis-retinal was characterized. In the NOCV framework, the formation of the sigma-bond appears as the sum of two complementary charge transfer processes from each vinyl fragment to the bond region, and partially to the other fragment. The formation of the pi-component of the bond is described by two pairs of NOCV representing the transfer of charge density from the neighboring 'double' C-C bonds. The NOCV eigenvalues and the related fragment-fragment bond multiplicities were used as quantitative measures of the sigma- and pi- contributions. The sigma-component of the 'single' C-C bonds appears to be practically constant in the systems analyzed, whereas the pi-contributions increase from butadiene (ca. 7.5%) to retinal (ca. 14%).