Alcoxyhalogenation d'acides gras ethyleniques. IV: Cas des octadécadienoates de méthyle 9c, 12c et 9t, 11t et de composés apparentés

Methoxybromination, with HBBT, of long chain methylene interrupted dienes led to simultaneous formation of methoxybromides and dimethoxydibromides. Formation of dimethoxydibromides in which methoxy groups are both internal is limited by steric hindrance.Methoxybromination of long chain conjugated dienes forms, in nearly equal quantities, methoxybromides resulting from a 1,4 addition and methoxybromides from 1,2 addition in which the methoxy group is adjacent to the double bond.     

Increasing Protein Stability by Polar Surface Residues: Domain-Wide Consequences of Interactions Within a Loop

We have examined the influence of surface hydrogen bonds on the stability of proteins by studying the effects of mutations of human immunoglobulin light chain variable domain (V_L). In addition to the variants Y27dD, N28F, and T94H of protein κIV Len that were previously described, we characterized mutants M4L, L27cN, L27cQ, and K39T, double mutant M4L/Y27dD, and triple mutant M4L/Y27dD/T94H. The triple mutant had an enhanced thermodynamic stability of 4.2 kcal/mol. We determined the structure of the triple mutant by x-ray diffraction and correlated the changes in stability due to the mutations with changes in the three-dimensional structure. Y27dD mutant had increased stability of Len by 2.7 kcal/mol, a large value for a single mutation. Asp27d present in CDR1 formed hydrogen bonds with the side-chain and main-chain atoms within the loop. In the case of the K39T mutant, which reduces stability by 2 kcal/mol, Lys39 in addition to forming a hydrogen bond with a carbonyl oxygen of a neighboring loop may also favorably influence the surface electrostatics of the molecule. We showed that hydrogen bonds between residues in surface loops can add to the overall stability of the V_L domains. The contribution to stability is further increased if the surface residue makes more than one hydrogen bond or if it forms a hydrogen bond between neighboring turns or loops separated from each other in the amino acid sequence. Based on our experiments we suggest that stabilization of proteins might be systematically accomplished by introducing additional hydrogen bonds on the surface. These substitutions are more straightforward to predict than core-packing interactions and can be selected to avoid affecting the protein’s function.     

Conformational change of the triple-helical structure. III. Stabilizing forces in the triple helix

The analysis of thermal melting curves of (PPG)_n (n = 10, 12, 14, and 15) and (PPG)_n(APG)_m (PPG)_n (2n + m = 15; m = 1, 3, and 5) revealed that the enthalpy and entropy changes accompanying the transition from the random coil to the triple helix are ?2500 cal and ?6.3 e.u. per one mole of the tripeptide of the form of Pro-Pro-Gly, and ?3100 cal and ?11.2 e.u. per one mole of the tripeptide of the form of Ala-Pro-Gly. The thermal instability of the triple helix composed of Ala-Pro-Gly sequences, compared to the helix of Pro-Pro-Gly sequences, is due to the larger entropy change of Ala-Pro-Gly (?11.2 e.u.) compared to that of Pro-Pro-Gly (?6.3 e.u.), not from the difference in the enthalpy change. The difference in the enthalpy change between Pro-Pro-Gly and Ala-Pro-Gly arises from the hydrophobic bond between two pyrrolidine rings of proline residues formed in the triple helix. Since the enthalpy change for the formation of hydrophobic bonds is positive, it is also concluded that only one hydrogen bond is formed in a tripeptide unit, regardless of the amino acid sequence. The enthalpy change for the formation of this hydrogen bond is ?3100 cal/mol, and that of the hydrophobic bond between two pyrrolidine rings is +600 cal/mol.     

The course of oxidative addition reactions of haloalkynes and haloalkenes to dimethyl- and dialkynylaurate(I) anions [RAuR]

The reactions of halo-alkynes Cl-CCH, C-lCC-Cl or PhCC-I with solutions of Li⁺[MeAuMe]⁻ in diethylether containing Ph₃P do not give the expected oxidative addition products Me₂(RCC)Au(PPh₃) with R = H, Cl, Ph. A mixture of other complexes is obtained instead which are generated in secondary reactions involving reductive elimination of ethane and/or dialkyne. However, addition of the halo-alkene H(Cl)CCCl₂ to the same substrate solution affords trans-Me₂[trans-H(Cl)CC(Cl)]Au(PPh₃) in good yield. Its molecular structure with pseudo-C_s symmetry has been determined by the solution NMR spectra and a single-crystal X-ray diffraction study. The reaction of methyl iodide with solutions of Li⁺[RCCAuCCR]⁻ in diethylether containing PPh₃ give the quaternary salts Ph₃PMe⁺ [RCCAuCCR]⁻ as the main products and only small amounts of cis-Me₂(RCC)Au(PPh₃) complexes probably formed in a series of oxidative addition, reductive elimination, and substitution reactions. The structure of Ph₃PMe⁺ [PhCCAuCCPh]⁻ has been determined.     

In situ proton NMR analysis of alpha-alkynoate biotransformations. From 'invisible' substrates to detectable metabolites.

Only 2% of the known natural products with acetylenic bonds are alpha-alkynoates. Their polarized, conjugated triple bond is an optimal target for an enzymic hydration. Therefore they are good substrates for the enzymes involved in metabolism of acetylenic compounds, resulting in products that are suitable for bacterial growth. We isolated a Pseudomonas putida strain growing on 2-butynedioate as well as on propynoate, and determined the metabolic pathways of these two alpha-alkynoates. The triple bonds in both compounds were initially hydrated and 2-ketobutandioate as well as 3-ketopropanoate were formed. These two beta-keto acids were decarboxylated resulting in pyruvate and acetaldehyde, respectively. Pyruvate was further hydrolysed mainly to acetate and formate, whereas minor amounts were reduced to lactate. In the other biotransformation, acetaldehyde was oxidized to acetate accompanied by the reduction of 3-ketopropanoate to 3-hydroxypropanoate. Analyses of these metabolic processes were performed by in situ 1H-NMR spectroscopy in 1H2O, although the substrates, propynoate and 2-butynedioate, carried only one or even no detectable protons, respectively. However, while protons from the solvent are incorporated in the course of the pathway, the metabolites can be detected and identified. Therefore a detailed determination of the metabolic process is possible.     

Effect of unsaturated bonds in the sn-2 acyl chain of phosphatidylcholine on the membrane-damaging action of Clostridium perfringens alpha-toxin toward liposomes

Clostridium perfringens alpha-toxin degrades phosphatidylcholine (PC) in the bilayer of liposomes and destroys the membrane. The effect of the type and position of unsaturation in the fatty acyl chain of PC (18:0/18:1 PC) synthesized on the toxin-induced leakage of carboxyfluorescein (CF) from PC liposomes was examined. Differential scanning calorimetry showed that the phase transition temperature (T_m) was minimal when the triple bond was positioned at C (9) in the sn-2 acyl chain. The toxin-induced CF leakage decreased with the migration of the bond from C (9) to either end of the acyl chain in PC. The PC containing the cis-double bond had a similar T_m to that with the triple bond, but a lower value than the PC containing the trans-double bond. Furthermore, the toxin-induced leakage from liposomes composed of PC containing the cis-double bond resembled that with PC having the triple bond and was greater than that from liposomes with PC having the trans-double bond. The binding of a H148G mutant to PC liposomes showed a reciprocal relationship in terms of the T_m value of PC containing the triple bond. These results indicate that the toxin-induced membrane damage is closely related to membrane fluidity in liposomes.     

A general synthesis of long chain ω- and (ω-1)-hydroxy fatty acids

A method for the synthesis of long chain fatty acids substituted at the ω and ω-1 positions has been developed. The key step is the isomerization of the triple bond of an alkyn-1-ol from an internal position in the chain to the free terminus with a new, convenient reagent, sodium aminopropylamide (NaAPA). Standard functional group manipulations i.e., Jones oxidation, esterification and hydroboration of the triple bond are used to prepare ω-hydroxy fatty esters. The generality of the method is illustrated with syntheses of ω-hydroxy fatty esters with 24, 26, 28 and 30 carbon chains.In the 24 carbon series, hydration of the terminal triple bond of alkynoic ester 4a followed by reduction gave the (ω-1)-hydroxy ester.     

The x ray structure of thiazolidine-4-carboxylic acid

The X ray crystal structure of thiazolidine-4-carboxylic acid has been determined. It appears that the anomalously low basicity of this compound in comparison with that of its non-sulfur-containing analogue, proline, is due to the resonance stabilization of the unprotonated form of thiazolidine-4-carboxylic acid. This stabilization is conferred through an increased interaction of the sulfur atom with the thiazolidine-4-carboxylic acid ring that is only possible when the nitrogen atom maintains a trigonal geometry. This effect seems also to account for the greater instability of N-hydroxymethylamines formed from thiazolidine-4-carboxylic acid as compared to those formed from proline. The crystal structure shows, in addition, an intramolecular hydrogen bond in combination with a bifurcated hydrogen bond.     

Role of hydrogen bond networks and dynamics in positive and negative cooperative stabilization of a protein

Cooperativity mediated through hydrogen bond networks in yeast iso-1-cytochrome c was studied using a thermodynamic triple mutant cycle. Three known stabilizing mutations, Asn 26 to His, Asn 52 to Ile, and Tyr 67 to Phe, were used to construct the triple mutant cycle. The side chain of His 26, a wild-type residue, forms two hydrogen bonds that bridge two substructures of the wild-type protein, and Tyr 67 and Asn 52 are part of an extensive buried hydrogen bond network. The stabilities of all variants in the triple mutant cycle were determined by guanidine hydrochloride denaturation methods and used to determine the pairwise, Delta(2)G(int), and triple interaction energies. His 26 and Ile 52 interact cooperatively (Delta(2)G(int) is 1-2 kcal/mol), whereas the two other pairs of mutations interact anticooperatively (Delta(2)G(int) is -0.5 to -1.5 kcal/mol). Previously reported structural data for iso-1-cytochrome c variants containing these mutations show that changes in the strength of the His 26 to Glu 44 hydrogen bond, apparently caused by changes in main chain dynamics, provide a mechanism for the long distance (His 26 to Phe 67 and His 26 to Ile 52) propagation of pairwise interaction energies. Opposing changes in the strength of the His 26 to Glu 44 hydrogen bond caused by the N52I and Y67F mutations generate a negative triple interaction energy (-0.9 +/-0.7 kcal/mol) that combined with cancellation of cooperative and anticooperative pairwise interactions produce apparent additivity for the stabilizing effects of the single mutations in the triple mutant variant.     

Amphiphilic properties of synthetic glycolipids based on amide linkages. I. Electron microscopic studies on aqueous gels

Amphiphiles with one or two amide linkages have been prepared by the reaction (A) of D-gluconic acid lactone with aliphatic amines (C₆-C₁₀) and (C) of N′-gluconoyl-ethylenediamine with alkanoic acids (C₆)-(C₁₀). Gel formation was found to occur on cooling the aqueous solutions at concentrations as low as 1–2%. Electron microscopy revealed that the gels of type A are composed of highly ordered ropes with right-handed twist, especially well developed with N-octylgluconamide. Type c substances with two amide linkages of opposite direction form gels consisting of smooth ribbons devoid of twisting. N-Methylation of the amide bond (type B and D substances) leads to a considerable increase in solubility. Gels are only formed from samples containing decanoic acid. These gels also consist of right-handed fibrillar ropes, only partially ordered with one N-methylated amide linkage (B), regularly aligned side-by-side with one N-methylated and one non-methylated amide bond (D). Gel formation and the typical morphology of the gels are discussed as arising mainly from strong intermolecular hydrogen bonds between amide linkages holding the molecules together and the influence of chiral centers of the carbohydrate chain which might be responsible for helical aggregates to be formed.     

Identification of 6-octadecynoic acid from a methanol extract of Marrubium vulgare L. as a peroxisome proliferator-activated receptor γ agonist

6-Octadecynoic acid (6-ODA), a fatty acid with a triple bond, was identified in the methanol extract of Marrubium vulgare L. as an agonist of peroxisome proliferator-activated receptor γ (PPARγ). Fibrogenesis caused by hepatic stellate cells is inhibited by PPARγ whose ligands are clinically used for the treatment of diabetes. Plant extracts of Marrubium vulgare L., were screened for activity to inhibit fibrosis in the hepatic stellate cell line HSC-T6 using Oil Red-O staining, which detects lipids that typically accumulate in quiescent hepatic stellate cells. A methanol extract with activity to stimulate accumulation of lipids was obtained. This extract was found to have PPARγ agonist activity using a luciferase reporter assay. After purification using several chromatographic methods, 6-ODA, a fatty acid with a triple bond, was identified as a candidate of PPARγ agonist. Synthesized 6-ODA and its derivative 9-octadecynoic acid (9-ODA), which both have a triple bond but in different positions, activated PPARγ in a luciferase reporter assay and increased lipid accumulation in 3T3-L1 adipocytes in a PPARγ-dependent manner. There is little information about the biological activity of fatty acids with a triple bond, and to our knowledge, this is the first report that 6-ODA and 9-ODA function as PPARγ agonists.     

A Highly Unusual Thioester Bond in a Pilus Adhesin Is Required for Efficient Host Cell Interaction

Many bacterial pathogens present adhesins at the tips of long macromolecular filaments known as pili that are often important virulence determinants. Very little is known about how pili presented by Gram-positive pathogens mediate host cell binding. The crystal structure of a pilus adhesin from the important human pathogen Streptococcus pyogenes reveals an internal thioester bond formed between the side chains of a cysteine and a glutamine residue. The presence of the thioester was verified using UV-visible spectroscopy and mass spectrometry. This unusual bond has only previously been observed in thioester domains of complement and complement-like proteins where it is used to form covalent attachment to target molecules. The structure also reveals two intramolecular isopeptide bonds, one of these formed through a Lys/Asp residue pair, which are strategically positioned to confer protein stability. Removal of the internal thioester by allele-replacement mutagenesis in S. pyogenes severely compromises bacterial adhesion to model host cells. Although current paradigms of bacterial/host cell interaction envisage strong non-covalent interactions, the present study suggests cell adhesion could also involve covalent bonds.     

Half-of-the-sites reactivity and all-of-the-sites substrate binding in transaldolase

Transaldolase from Candida utilis is a dimeric protein composed of two identical subunits. The cleavage of fructose 6-phosphate by this enzyme was followed in a rapidmixing spectrophotometer. A very rapid reaction was observed during which 1 mol of glyceraldehyde 3-phosphate/mol of enzyme was released, followed by a much slower reaction in which additional glyceraldehyde 3-phosphate was formed. Binding studies carried out with the same substrate showed that two equivalents of dihydroxyacetone were bound. These results indicate that both sites are active, but that only one functions in the rapid catalytic reaction. The half-of-the-sites reactivity of transaldolase may be attributed to a high degree of negative cooperativity between the two subunits.     

Covalent heme attachment to the protein in human heme oxygenase-1 with selenocysteine replacing the His25 proximal iron ligand

To characterize heme oxygenase with a selenocysteine (SeCys) as the proximal iron ligand, we have expressed truncated human heme oxygenase-1 (hHO-1) His25Cys, in which Cys-25 is the only cysteine, in the Escherichia coli cysteine auxotroph strain BL21(DE3)cys. Selenocysteine incorporation into the protein was demonstrated by both intact protein mass measurement and mass spectrometric identification of the selenocysteine-containing tryptic peptide. One selenocysteine was incorporated into approximately 95% of the expressed protein. Formation of an adduct with Ellman’s reagent (DTNB) indicated that the selenocysteine in the expressed protein was in the reduced state. The heme-His25SeCys hHO-1 complex could be prepared by either (a) supplementing the overexpression medium with heme, or (b) reconstituting the purified apoprotein with heme. Under reducing conditions in the presence of imidazole, a covalent bond is formed by addition of the selenocysteine residue to one of the heme vinyl groups. No covalent bond is formed when the heme is replaced by mesoheme, in which the vinyls are replaced by ethyl groups. These results, together with our earlier demonstration that external selenolate ligands can transfer an electron to the iron [Y. Jiang, P.R. Ortiz de Montellano, Inorg. Chem. 47 (2008) 3480-3482 ], indicate that a selenyl radical is formed in the hHO-1 His25SeCys mutant that adds to a heme vinyl group.     

Alkyl isocyanates as active site-directed inactivators of guinea pig liver transglutaminase.

Alkyl isocyanates are effective inactivators of guinea pig liver transglutaminase. Based on the specificity of the reaction the protection against inactivation by glutamine substrate, and the essential nature of calcium for the inactivation reaction, it is concluded that these reagents act as amide substrate analogs and, thus function in an active site-specific manner. Support for the contention that inactivation results from alkyl thiocarbamate ester formation through the single active site sulfhydryl group of the enzyme is (a) the loss of one free--SH group and the incorporation of 1 mol of reagent/mol of enzyme in the reaction, (b) similarity in chemical properties of the inactive enzyme derivative formed to those previously reported for another alkyl thiocarbamoylenzyme and an alkyl thiocarbamoylcysteine derivative, and (c) the finding that labeled peptides from digests of [methyl-14C]thiocarbamoyltransglutaminase and those from digests of iodoacetamide-inactivated enzyme occupy similar positions on peptide maps. Transglutaminase was found to be inactivated neither by urethan anlogs of its active ester substrates nor by urea analogs of its amide substrates. It is concluded on the basis of these findings that inactive carbamoylenzyme derivatives are formed only by direct addition of the transglutaminase active--SH group to the isocyanate C--N double bond, and not, like several serine active site enzymes, by nucleophilic displacement with urethan analogs of substrate, or by nucleophilic displacement with urea analogs of substrate.     

The crystal structures of NSF3 and (NSF2N(CH3)CH2-)2: How short is the ‘Crystallographic’ NS triple bond?

The synthesis of the first unequivocally characterised bis(difluorothiazyne), [NSF₂N(CH₃)CH₂-]₂ is reported. The crystal structures of this and NSF₃ are also reported. NSF₃ has the same geometrical parameters, within error, as it does in the gas phase. PIXEL calculations show that the principal interactions in its crystal structure are SN?SN dipolar contacts, which form chains with S?N = 3.533(2) Å. These contacts are reminiscent of those observed in the crystal structures of ketones. The exchange of a fluorine by a dialkylamino group has almost no influence on the NS bond distance while the SF bonds are significantly elongated. This behaviour is explained by negative hyperconjugation and confirmed by experimental data (as far as available) and quantum chemical calculations for NSF_n(NMe₂)_3−n and NSF_nPh_3−n (n = 1-3).     

Structure-Activity Relationships of Abscisic Acid Analogs Based on the Induction of Freezing Tolerance in Bromegrass (Bromus inermis Leyss) Cell Cultures

The induction of freezing tolerance in bromegrass (Bromus inermis Leyss) cell culture was used to investigate the activity of absisic acid (ABA) analogs. Analogs were either part of an array of 32 derived from systematic alterations to four regions of the ABA molecule or related, pure optical isomers. Alterations were made to the functional group at C-1 (acid replaced with methyl ester, aldehyde, or alcohol), the configuration at C-2, C-3 (cis double bond replaced with trans double bond), the bond order at C-4, C-5 (trans double bond replaced with a triple bond), and ring saturation (C-2′, C-3′ double bond replaced with a single bond so that the C-2′ methyl and side chain were cis). All deviations in structure from ABA reduced activity. A cis C-2, C-3 double bond was the only substituent absolutely required for activity. Overall, acids and esters were more active than aldehydes and alcohols, cyclohexenones were more active than cyclohexanones, and dienoic and acetylenic analogs were equally active. The activity associated with any one substituent was, however, markedly influenced by the presence of other substituents. cis, trans analogs were more active than their corresponding acetylenic analogs unless the C-1 was an ester. Cyclohexenones were more active than cyclohexanones regardless of oxidation level at C-1. An acetylenic side chain decreased the activity of cyclohexenones but increased the activity of cyclohexanones relative to their cis, trans counterparts. Trends suggested that for activity the configuration at C-1′ has to be the same as in (S)-ABA, in dihydro analogs the C-2′-methyl and the side chain must be cis, small positional changes of the 7′-methyl are tolerable, and the C-1 has to be at the acid oxidation level.     

Crystal and molecular structures of {Cu(μ-CCPh)(triphos)}2 [triphos = (PPh2CH2)3CMe]

The binuclear complex {Cu(μ-CCPh)(triphos)}₂ [triphos = (PPh₂CH₂)₃CMe] has been obtained from a reaction between {Cu(CCPh)}_n and triphos. The two copper atoms are bridged unsymmetrically by two CCPh groups, each attached through one carbon only [Cu-C, 2.016(4) Å], the separation between the two coppers being 2.4663(8) Å. Only two of the three phosphorus atoms in each ligand are coordinated to copper [Cu-P(1,2) 2.281, 2.273(1) Å]. The observed structure may be rationalised using a recent theoretical study [C. Mealli, S.S.M.C. Godinho, M.J. Calhorda, Organometallics 20 (2001) 1734] and differs from that assumed for the rationalisation of its luminescence properties [V. Pawlowski, G. Knör, C. Lennartz, A. Vogler, Eur. J. Inorg. Chem. (2005) 3167].