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Biosynthesis, isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration.

ABSTRACT: Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.

SUBMITTER: Jin J

PROVIDER: S-EPMC3617949 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Catalytic activities of mammalian epoxide hydrolases with cis and trans fatty acid epoxides relevant to skin barrier function.

Project description:Lipoxygenase (LOX)-catalyzed oxidation of the essential fatty acid, linoleate, represents a vital step in construction of the mammalian epidermal permeability barrier. Analysis of epidermal lipids indicates that linoleate is converted to a trihydroxy derivative by hydrolysis of an epoxy-hydroxy precursor. We evaluated different epoxide hydrolase (EH) enzymes in the hydrolysis of skin-relevant fatty acid epoxides and compared the products to those of acid-catalyzed hydrolysis. In the absence of enzyme, exposure to pH 5 or pH 6 at 37°C for 30 min hydrolyzed fatty acid allylic epoxyalcohols to four trihydroxy products. By contrast, human soluble EH [sEH (EPHX2)] and human or murine epoxide hydrolase-3 [EH3 (EPHX3)] hydrolyzed cis or trans allylic epoxides to single diastereomers, identical to the major isomers detected in epidermis. Microsomal EH [mEH (EPHX1)] was inactive with these substrates. At low substrate concentrations (<10 ?M), EPHX2 hydrolyzed 14,15-epoxyeicosatrienoic acid (EET) at twice the rate of the epidermal epoxyalcohol, 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoic acid, whereas human or murine EPHX3 hydrolyzed the allylic epoxyalcohol at 31-fold and 39-fold higher rates, respectively. These data implicate the activities of EPHX2 and EPHX3 in production of the linoleate triols detected as end products of the 12R-LOX pathway in the epidermis and implicate their functioning in formation of the mammalian water permeability barrier.

| S-EPMC5880498 | biostudies-literature

Influence of the cis/trans configuration on the supramolecular aggregation of aryltriazoles.

Project description:The ability of trans- and cis-1,2-glucopyranosyl and cyclohexyl ditriazoles, synthesized by CuAAC "click" chemistry, to form gels was studied, their physical properties determined, and the self-aggregation behavior investigated by SEM, X-ray, and EDC studies. The results revealed that self-assembly was driven mainly by ?-? stacking interactions, in addition to hydrogen bonding, with the aromatic rings adopting a high degree of parallelism, as seen in crystal packings and ECD data. Furthermore, ?-bromine interactions between the bromine atom of the aryl substituents and the triazole units might also contribute to an overall stabilization of the supramolecular aggregation of bis(4-bromophenyl)triazoles. The trans or cis spatial disposition of the triazole rings is highly important for gelation, with the cis configuration having higher propensity.

| S-EPMC6902786 | biostudies-literature

Azomethine ylide mediated inversion of configuration of quaternary imidazoline carbon: converting trans- to its cis- imidazolines.

Project description:An efficient synthetic methodology of converting trans-4,5-diaryl-2-imidazolines to the corresponding cis-4,5-diaryl-2-imidazolines has been developed. This methodology features mild reaction conditions and a simple one-pot two-step procedure.

| S-EPMC3157035 | biostudies-literature

Cis-trans

Project description:Cis-trans

| PRJEB1227 | ENA

Probing Peptidylprolyl Bond cis/trans Status Using Distal <sup>19</sup> F NMR Reporters.

Project description:A method for measuring peptidylprolyl bond cis-trans conformational status in peptide models is described, using 4-fluorophenylalanine (4FPhe) as a distal reporter for 19 F NMR. The %cis-Pro population was measured for peptides of the general structure Ac-X-Pro-Z-Ala-Ala-4FPhe (X and Z are proteinogenic amino acids) at pH 7.4, and provided conformational populations consistent with literature values obtained by more complex methods. This approach was applied to probe the prolyl bond status in pentapeptide models of the intrinsically disordered C-terminal region of α-synuclein, which mirrored the preferences in the Ac-X-Pro-Z-Ala-4FPhe models. Advantageously, the 19 F reporter group does not need to be adjacent to or attached to proline to provide quantifiable signals and distal 4-fluorophenylalanines can be placed so as not to influence prolyl bond conformation. Finally, we demonstrated that the prolyl bond status is not significantly affected by pH when there are ionisable amino acid residues at the carboxyl side of proline, which makes 19 F NMR an invaluable tool with which to study proline isomerism at a range of pHs and in different solvents and buffers.

| S-EPMC10946801 | biostudies-literature

Cis-trans

Project description:Differential Expression analysis in Drosophila pseudoobscura to identify cis-trans regulation

2014-03-27 | E-MTAB-1424 | biostudies-arrayexpress

Does the cis/trans configuration of peptide bonds in bioactive tripeptides play a role in ACE-1 enzyme inhibition?

Project description:BackgroundThe milk casein-derived bioactive tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) have been shown to prevent development of hypertension in animal models and to lower blood pressure in moderately hypertensive subjects in most but not all clinical trials. Inhibition of angiotensin-converting enzyme 1 (ACE-1) has been suggested as the explanation for these antihypertensive and beneficial vascular effects. Previously, human umbilical vein endothelial cells (HUVEC) have not been used to test ACE-1 inhibiting properties of casein derived tripeptides in vasculature.PurposeWe focused on the cis/trans configurations of the peptide bonds in proline-containing tripeptides in order to discover whether the different structural properties of these peptides influence their activity in ACE-1 inhibition. We hypothesized that the configuration of proline-containing peptides plays a significant role in enzyme inhibition.MethodsAutoDock 4.2 docking software was used to predict suitable peptide bond configurations of the tripeptides. Besides modeling studies, we completed ACE-1 activity measurements in vitro using HUVEC cultures.ResultsIn HUVEC cells, both IPP and VPP inhibited ACE-1. Based on molecular docking studies, we propose that in ACE-1 inhibition IPP and VPP share a similar cis configuration between the first aliphatic (isoleucine or valine) and the second (proline) amino acid residues and more different configurations between two proline residues. In vivo experiments are needed to validate the significance of the present findings.

| S-EPMC3930482 | biostudies-literature

Cis/Trans Energetics in Epoxide, Thiirane, Aziridine and Phosphirane Containing Cyclopentanols: Effects of Intramolecular OH⋯O, S, N and P Contacts.

Project description:A recent computational analysis of the stabilizing intramolecular OH⋯O contact in 1,2-dialkyl-2,3-epoxycyclopentanol diastereomers has been extended to thiiriane, aziridine and phosphirane analogues. Density functional theory (DFT), second-order Møller-Plesset perturbation theory (MP2) and CCSD(T) coupled-cluster computations with simple methyl and ethyl substituents indicate that electronic energies of the c i s isomers are lowered by roughly 3 to 4 kcal mol-1 when the OH group of these cyclopentanol systems forms an intramolecular contact with the O, S, N or P atom on the adjacent carbon. The results also suggest that S and P can participate in these stabilizing intramolecular interactions as effectively as O and N in constrained molecular environments. The stabilizing intramolecular OH⋯O, OH⋯S, OH⋯N and OH⋯P contacts also increase the covalent OH bond length and significantly decrease the OH stretching vibrational frequency in every system with shifts typically on the order of -41 cm-1.

| S-EPMC6680949 | biostudies-literature

Density Functional Theory based study on structural, vibrational and NMR properties of cis - trans fulleropyrrolidine mono-adducts.

Project description:Since the early nineties, countless publications have been devoted to the study of possible uses of [60] fullerene (C60) and its derivatives in the fields of materials and nano-biomedical sciences. However, in spite of the importance of conformers notably from the pharmacological point of view, the cis/trans isomerization of C60 mono-adducts has been rarely seldom investigated. Here we present the results of DFT calculations of the structural, vibrational and NMR properties of both cis and trans isomers of fulleropyrrolidine mono-adduct obtained by photo-addition of glycine methyl ester to C60. Taken together, our results have shown that the cis isomer is more stable than the trans one. For the cis conformation, the simulated vibrational spectrum shows a more intense peak at 1298 cm-1. While 13C spectra revealed no significant differences between the two isomers as compared to experimental results, the calculated 1H chemical shifts show a significant difference between the two conformers in both the gas phase and in solution. The trans isomer presents a proton at 5.86 ppm, which is more deshielded than the proton of the cis conformer (5.24 ppm).

| S-EPMC6242360 | biostudies-literature

Leukotriene B4 Is a Major Determinant of Leukocyte Recruitment During Otitis Media.

Project description:BackgroundPathogens of otitis media (OM) induce inflammatory responses in the middle ear (ME), characterized by mucosal hyperplasia, leukocyte infiltration, and inflammatory mediators, including arachidonic acid metabolites. We studied the role of the eicosanoid leukotriene B4 (LTB4) in OM.MethodsExpression of LTB4-related genes was evaluated by gene array and single-cell RNA-Seq in MEs infected with nontypeable Haemophilus influenzae (NTHi). An inhibitor of LTB4 receptor 1 (i.e. U75302) was also used to block LTB4 responses.ResultsME expression of LTB4-related genes was observed by gene arrays and scRNA-Seq. However, not all genes involved in LTB4 generation occurred in any one specific cell type. Moreover, LTB4 receptor inhibition significantly reduced mucosal hyperplasia and virtually eliminated leukocyte infiltration.ConclusionsME expression of LTB4-related genes suggest a functional role in OM disease. The fact that LTB4-generation is spread across different cell types is consistent with a transcellular pathway of eicosanoid biosynthesis involving cell-to-cell signaling as well as transfer of biosynthetic intermediates between cells. The dramatic reduction in ME leukocyte infiltration caused by U75302 indicates that LTB4 plays a major role in ME inflammatory cell recruitment, acting via the LTB4R1 receptor. Given that there are many other chemotactic factors that occur in the ME during OM, the ability of LTB4 to activate leukocytes and stimulate their extravasation may explain the effects of inhibition. Reduction in mucosal hyperplasia due to U75302 administration may be secondary to the reduction in leukocytes since LTB4R1 is not expressed by mucosal epithelial or stromal cells. The results suggest that LTB4 receptor antagonists could be useful in treating OM.

| S-EPMC8727869 | biostudies-literature

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