Partial purification and properties of a cis-3: trans-2-enal isomerase from cucumber fruit

An enzyme, which catalyses the isomerisation of cis-3-enals to trans-2-enals, has been partially purified from cucumber fruit. The isomerase activity has been resolved from significant contamination by the related activities, lipoxygenase and hydroperoxide cleavage enzymes. An examination of the substrate specificity of the isomerase enzyme showed it to be specific for the cis-3-enals. The most efficient isomerisation was achieved with cis-3-hexenal and cis-3-nonenal which are, physiologically, the two most significant substrates. The trans-3-enal and cis-3-enol were not suitable substrates for the enzyme.     

Preparation of methyl cis-9,cis-12-octadecadienoate-16,16,17,17-d4

An eight-step synthesis is described which gives an overall yield of ～30% methyl cis-9,cis-12-octadecadienoate-16,16,17,17-d₄. The preparation utilizes easily obtainable starting materials. Tris(triphenylphosphine)chlororhodium (I) catalyst is used for incorporation of the deuterium isotopes. The double bond in the 9 position is created by the Wittig coupling of 1-non-3-enyl-d₄-triphenylphosphonium bromide to methyl 8-formyloctanoate. Various methods for preparation of the intermediate and final products are discussed. Partial argentation resin chromatography was used to remove the ～9% trans/cis, cis/trans, and trans/trans isomers also produced. Analysis of the final product by mass spectrometry (MS) indicated 96%-d₄.     

Different reaction mechanisms for cis- and trans-prenyltransferases

Octaprenyl diphosphate synthase (OPPs) and undecaprenyl diphosphate synthases (UPPs) catalyze consecutive condensation reactions of farnesyl diphosphate (FPP) with 5 and 8 isopentenyl diphosphate (IPP) to generate C₄₀ and C₅₅ products with trans- and cis-double bonds, respectively. In this study, we used IPP analogue, 3-bromo-3-butenyl diphosphate (Br-IPP), in conjunction with radiolabeled FPP, to probe the reaction mechanisms of the two prenyltransferases. Using this alternative substrate with electron-withdrawing bromo group at the C3 position to slow down the condensation step, trapping of farnesol in the OPPs reaction from radiolabeled FPP under basic condition was observed, consistent with a sequential mechanism. In contrast, UPPs reaction yielded no farnesyl carbocation intermediate under the same condition with radiolabeled FPP and Br-IPP, indicating a concerted mechanism. Our data demonstrate the different reaction mechanisms for cis- and tran-prenyltransferases although they share the same substrates.     

Effect of cis-9,trans-11 and trans-10,cis-12 isomers of conjugated linoleic acid on the integrity and functionality of cryopreserved bovine spermatozoa

Plasma membranes of sperm subjected to low temperatures undergo changes in their structure and permeability. The addition of fatty acids in semen cryopreservation media may influence the sperm motility after thawing, possibly by maintaining the membrane fluidity due to their incorporation in lipid bilayers. In this work, different concentrations of the isomers cis-9,trans-11 and trans-10,cis-12 of conjugated linoleic acid (CLA) were added in the cryopreservation medium of bovine sperm. Four Jersey bulls were used, and the ejaculates were processed as a pool. The Tris-based extender (Dilutris®) was supplemented with 20% egg yolk (MB). The treatments with CLA (Luta-CLA®), which had oily presentation, were prepared from MB with addition of 1% sodium lauryl sulfate, and denominated MBL. The concentrations of CLA tested were 50, 100, and 150 μM. The motility characteristics of the post-thaw semen were analyzed by computerized analysis system (CASA), and plasma membrane integrity and acrosomal and mitochondrial function assessed by the association of the fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), JC-1 and Hoechst 33342. No significant differences were observed among treatments, excepting for a decreased mitochondrial potential of cells treated with 150 μM CLA. The addition of CLA, at the concentrations used, showed no advantages on the integrity and functionality of bovine sperm submitted to cryopreservation.     

Identification of cis-9,10-methylenehexadecanoic acid in submitochondrial particles of bovine heart

Submitochondrial particles of bovine heart were hydrolyzed by phospholipase A₂ and the products were analyzed by liquid chromatography electrospray ionization-mass spectrometry. We found a fatty acid with a molecular mass of 268 Da and a retention time longer than that of linoleic acid. Next, we synthesized organically cis-9,10-methylenehexadecanoic acid, which has a molecular mass similar to that of the extracted fatty acid, and characterized its high performance liquid chromatography and gas chromatography-mass spectrometry profiles. Using these data we were able to identify endogenous cis-9,10-methylenehexadecanoic acid in rat and human heart and liver tissues that had been hydrolyzed by phospholipase A₂. This fatty acid was not detected in tissue extracts that had not been hydrolyzed by phospholipase A₂. Similar amounts of cis-9,10-methylenehexadecanoic acid were measured in tissue extracts after total hydrolysis. These results suggest that cis-9,10-methylenehexadecanoic acid is a fatty acid component, in the sn-2 position, of phospholipids in some mammalian tissue.     

Enhancement of antibody synthesis in rats by feeding cis-9,trans-11 conjugated linoleic acid during early life

Previous studies have demonstrated that the intake of a 1% conjugated linoleic acid (CLA) diet in an 80:20 mixture of cis-9,trans-11 and trans-10,cis-12 exerts age-specific effects on the immune system: immunoglobulin enhancement and proliferative down-modulation in neonatal and adult rats, respectively. The present study evaluates the influence of the same diet on antibody synthesis of early infant Wistar rats during suckling and/or after weaning. Dietary supplementation was performed during suckling and early infancy (4 weeks), only during suckling (3 weeks), or only in early infancy (1 week). CLA content in plasma and serum immunoglobulin (Ig) G, IgM and IgA concentration were determined. Proliferation, cytokines and Ig production were evaluated on isolated splenocytes. Cis-9,trans-11- and trans-10,cis-12-CLA isomers were detected in the plasma of all CLA-supplemented animals, and the highest content was quantified in those rats supplemented over the longest period. These rats also exhibited higher concentrations of serum IgG, IgM and IgA. Moreover, splenocytes from CLA-supplemented rats showed the highest IgM and IgG synthesis and interleukin (IL)-6 production, whereas their proliferative ability was lower. In summary, in infant rats, we observed both the enhance antibody synthesis previously reported in neonates, and the reduced lymphoproliferation previously reported in adults.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

Synthesis, characterization and kinetic studies of the cis-[RuCl2(cyclen)] complex

We report here the synthesis, characterization and kinetic studies of cis-[RuCl₂(cyclen)]⁺ in aqueous solution, where cyclen is the macrocyclic ligand 1,4,7,10-tetraazacyclododecane. The complex releases one Cl⁻ producing cis-[RuCl(OH)(cyclen)]⁺ in aqueous solution at pH 4.60. The product of this reaction was characterized by Ultraviolet-Visible (UV-Vis) spectrum in comparison to the synthesized cis-[RuCl(OH)(cyclen)](BF₄)·2H₂O. The electrochemical data showed that E_pc of the Ru(III/II) peak increases as the macrocycle ring size decreases and also when the trans conformation is changed to cis. The chloride affinity of Ru(III) depends on the macrocycle ring size since cis-[RuCl₂(cyclam)]⁺ (cyclam=1,4,8,11-tetraazacyclotetradecane) does not release chloride for at least 12 h. The overall effect between cyclam and cyclen reflects the fact that the electron involved in the reduction enters a nonbonding π-d orbital and its energy is affected by the macrocyclic ligand.     

cis- and trans-phytoene in Verticillium agaricinum

cis-Phytoene and trans-phytofluene were identified in illuminated cultures of Verticillium agaricinum in addition to the other carotenoids     

Synthesis of new chiral cis-3-aminoazetidines and their use in catalytic asymmetric reactions

A new series of chiral cis-3-aminoazetidines have been prepared from (S)-1-phenylethylamine. The catalytic activity of the new ligands has been tested in standard asymmetric reactions, in most cases moderate to good yields and moderate enantioselectivity have been observed.     

Complex of cis-(NH3)2PtCl2 with guanylyl(3′-5′)-cytidine

The predominant complex formed by the reaction of cis-(NH₃)₂PtCl₂ and guanylyl(3′-5′)cytidine has been isolated. The molar ratio of the binding of cis-(NH₃)₂PtCl₂ to guanylyl(3′-5′)-cytidine is 1:2. The values of proton dissociation constant due to guanine and cytosine bases provide useful information for determining the binding site of the isolated complex. In addition, nmr and ir spectral data were used to determine the binding site. cis-(NH₃)₂PtCl₂ coordinates to guanylyl(3′-5′)cytidine through N(7) position of the guanine base, but cytosine base does not participate in the binding to cis-(NH₃)₂Pt²⁺. Interbase crosslink has not been detected. The binding specificity of cis-(NH₃)₂PtCl₂ to guanine base is discussed.     

2,4-Trans-4,5-trans-4,5-dihydroxypipecolic acid and cis-5-hydroxypipecolic acid from leaves of Calliandra angustifolia and sap of C. Confusa

2,4-Trans-4,5-trans-4,5-dihydroxypipecolic acid and cis-5-hydroxypipecolic acid have been isolated from the leaves of Calliandra angustifolia and the sap of C. confusa. Distribution of these and other non-protein amino acids is discussed.     

Increase of cis-3-hexen-1-ol content in tea leaves following mechanical injury

Dark-grown tea seedlings were exposed to ¹⁴CO₂ and incubated for 1 month. From a comparison of the radioactivities in lipid fractions obtained from both intact green seedlings and mechanically injured seedlings, it was found that the radioactivity in cis-3-hexen-1-ol was significantly increased by mechanical injury.     

Cis-4-hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid from Calliandra

cis-4-Hydroxypipecolic acid and 2,4-cis-4,5-trans-4,5-dihydroxypipecolic acid were isolated from leaves of Calliandra pittieri. A system for resolving the eight imino acids isolated from Calliandra is described.     

Solvent exchange, solvent interchange, aquation and isomerisation reactions of cis- and trans-[Co(tmen)2(NCMe)2] in water, Me2SO and MeCN: kinetics and stereochemistry

The synthesis and characterisation of cis- and trans-[Co(tmen)₂(NCCH₃)₂](ClO₄)₃ are described. Solvolysis rates have been measured by both ¹H NMR spectroscopy and UV-Vis spectrophotometry in dimethyl sulfoxide at 298.2 K. The cis isomer undergoes solvolysis by consecutive first-order reactions, k₁=5.61 × 10⁻⁴ and k₂=5.35 × 10⁻⁴ s⁻¹, each with steric retention. The measured solvolysis rate (single step reaction) for the trans isomer is k=1.54 × 10⁻⁵ s⁻¹. The solvent exchange rates have been measured by ¹H NMR spectroscopy in CD₃CN at 298.2 K: k_ex(cis)=k_ct + k_cc=2.0 × 10⁻⁵ and k_ex(trans)=k_tc + k_tt=4.56 × 10⁻⁶ s⁻¹. From these data, the measured cis-trans isomerisation rate (1.71 × 10⁻⁶ s⁻¹) and equilibrium position in CH₃CN (17% trans), the steric course for substitution in the exchange processes has been determined: trans reactant - 69% trans product; cis reactant - 99% cis product. Aquation rates for cis- and trans-[Co(tmen)₂(NCCH₃)₂](ClO₄)₃ have also been determined spectrophotometrically and by NMR; k_cis=1.3 × 10⁻⁴ and k_trans=2.7 × 10⁻⁵ s⁻¹. In both cases the steric course for the primary aquation step is indeterminate because the subsequent steps are faster. Where data are available, the [Co(tmen)₂X₂]ⁿ⁺ complexes are found to be consistently much more reactive than their [Co(en)₂X₂]ⁿ⁺ analogues.     

The differential effects produced by cis- and trans-DDP on DNA in calf thymus nucleosomes

Calf thymus nucleosomes containing H1 were treated with dichlorodiammineplatinum (DDP) at low binding ratios (r = 0.05–0.15). Change in the electrophoretic mobility of the extracted nucleosomal DNA was observed following treatment with cis-DDP and little change with trans-DDP. There was a decrease in the electrophoretic mobility of the nucleosomal DNA as well as obliteration of the nucleosomal repeat distance. The fluorescence intensity of terbium binding to the extracted DNA showed minimal change following drug treatment. However, the thermal melting behavior of the nucleosomal DNA was altered to a greater extent following cis-DDP treatment at 280 rather than 260 nm and a destabilization of the DNA helix was observed. These data suggest that in the whole nucleosome, cis-DDP produces greater structural effects on the packaged DNA than trans-DDP, although similar amounts of drug are bound with both isomers.     

trans- and cis-Ru(II) aminophosphine complexes: Syntheses, X-ray structures and catalytic activity in transfer hydrogenation of acetophenone derivatives

The ability of transition metal catalysts to add or remove hydrogen from organic substrates by transfer hydrogenation process is a valuable synthetic tool. For this aim, a novel Ru(II) complex with the P-N ligand [(Ph₂P)₂NCH₂-C₄H₃S] derived from thiophene-2-methylamine was synthesized starting with the complex [Ru(η⁶-p-cymene)(μ-Cl)Cl]₂ and isolated in two isomeric forms: trans- and cis-[Ru((PPh₂)₂NCH₂-C₄H₃S)₂Cl₂], 2 and 3, respectively. The structures of both isomers were also determined by single crystal X-ray diffraction. The cis-isomer 3 can be isolated from the solution of major trans-isomer 2 as yellow crystals. However, upon dissolution 3 is rapidly converted to the trans-isomer 2. The new ruthenium(II) complex provides high catalytic activity in the transfer hydrogenation of acetophenone derivatives to 1-phenylethanol derivatives in the presence of 2-propanol as the hydrogen source. This transfer hydrogenation is characterized by low reversibility under the experimental conditions.     

Possible chemotaxonomic significance of the occurrence of cis-4-hydroxy-l-proline in santalaceae

cis-4-hydroxy-l-porline was detected in three genera (four species) of Santalaceae.     

Multinuclear NMR study and crystal structures of complexes of the types cis- and trans-Pt(amine)2I2

Complexes of the types cis- and trans-Pt(amine)₂I₂ were studied by spectroscopic methods, especially by multinuclear NMR spectroscopy. In ¹⁹⁵Pt NMR, the cis diiodo compounds with primary amines were observed between −3342 and −3357 ppm in acetone, while the trans compounds were found between −3336 and −3372 ppm. For the secondary amines, the chemical shifts were observed at lower fields. In ¹H NMR, the trans complexes were observed at higher fields than the cis compounds, while in ¹³C NMR, the reverse was observed. The ²J(¹⁹⁵Pt-¹H) and ³J(¹⁹⁵Pt-¹H) coupling constants are larger for the cis compounds (ave. 67 and 45 Hz, respectively) than for the trans isomers (ave. 59 and 38 Hz). In ¹³C NMR, the values of ²J(¹⁹⁵Pt-¹³C) and ³J(¹⁹⁵Pt-¹³C) were also found to be larger for the cis complexes (ave. 17 and 39 Hz versus 11 and 28 Hz). There seems to be a slight dependence of the pK_a values of the protonated amines or the proton affinity in the gas phase with the δ(Pt) chemical shifts. The crystal structures of eight diiodo complexes were determined. These compounds are cis-Pt(CH₃NH₂)₂I₂, cis-Pt(n-C₄H₉NH₂)₂I₂, cis-Pt(Et₂NH)₂I₂, trans-Pt(n-C₃H₇NH₂)₂I₂, trans-Pt(iso-C₃H₇NH₂)₂I₂, trans-Pt(n-C₄H₉NH₂)₂I₂, trans-Pt(t-C₄H₉NH₂)₂I₂ and trans-Pt(Me₂NH)₂I₂. The Pt-N bond distances located in trans position to the iodo ligands were compared to those located in trans position to the amines. The Pt-N bond in cis-Pt(Et₂NH)₂I₂ are much longer than the others, probably caused by the steric hindrance of the two very bulky ligands located in cis positions.