Synthesis of site-specifically labeled arachidonic acids as mechanistic probes for prostaglandin H synthase

[structure: see text] Prostaglandin H synthase catalyzes the first committed step in the biosynthesis of prostaglandins and thromboxane. Herein we report the synthesis of four site-specifically labeled arachidonic acids for investigation of the radical intermediate formed during this enzymatic reaction. Two compounds were prepared using a common C9-C11 fragment, while another target was synthesized using a previously reported advanced intermediate. An alkyne coupling followed by hydrogenation and Wittig reaction was used to prepare the final labeled substrate.     

Determination of arachidonic acid based on the prostaglandin H synthase catalyzed reaction

This article presents a novel method of arachidonic acid (AA) determination based on the reaction catalyzed by prostaglandin H synthase (PGHs). The deoxygenated and nondeoxygenated (as control) buffers were used to obtain the PGHs preparations from bovine vesicular glands by two different methods. The higher specific activity was observed for solubilized preparations obtained by ultracentrifugation and deoxygenated buffers. The preparations obtained by Ca²⁺ treatment demonstrated higher stability of PGHs during its storage at −15°C. To record the initial rate of AA transformation, a spectrophotometric assay of PGHs cyclo-oxygenase and peroxidase activities was developed using adrenaline and ABTS as electron donors. No oxidation of A BTS was observed in the reaction of AA transformation catalyzed by the PGHs from bovinevesicular glands. However, this electron donor was successfully used in the reaction catalyzed by PGHs from sheep vesicular glands. No chemilum inescence was recorded in the reaction of AA transformation catalyzed by PGHs from bovine vesicular glands in the presence of luminol. The chemiluminescent intensity was measured after addition of hydrogen peroxide allowing quantitative assay of AA to be performed.     

Proteome analysis using selective incorporation of isotopically labeled amino acids

A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of this method for unambiguously identifying proteins isolated from E. coli.     

Synthesis and characterization of a new prostaglandin H synthase model

Prostaglandin H synthases (PGHSs) are heme-containing enzymes, which directly add two O₂ molecules to arachidonic acid (AA) by the initiation of H-atom abstraction of 1,4-diene moiety and through sequential reactions to give PGH₂ as a final product. Here, we report the synthesis of a new PGHS model, which has two binaphthol bridges in one side of an iron porphyrin. Its substrate reaction site is more flexible and accordingly less hindered than the previous four binaphthol-bridged twin-coronet-type models (FeTCP). The present model is expected to accept higher olefins, which can reach the intermediate phenoxyl radical and iron in this complex. Upon treatment of the iron complex with mCPBA at −50 °C, we succeeded in the quantitative generation of the corresponding naphthoxyl radical, which was characterized by the increase in characteristic absorbance for naphthoxyl radical at 385 and 485 nm as well as by its ESR signal and recovery of the original spectra by the addition of N,N-dimethylaniline as an efficient reducing agent.     

A theoretical study of radical-only and combined radical/carbocationic mechanisms of arachidonic acid cyclooxygenation by prostaglandin H synthase

Prostaglandin H synthase catalyzes the oxygenation of arachidonic acid into the cyclic endoperoxide, prostaglandin G₂ (PGG₂), and the subsequent reduction of PGG₂ to the corresponding alcohol, prostaglandin H₂ (PGH₂), the precursor of all prostaglandins and thromboxanes. Both radical abstraction by a neighboring tyrosyl radical and combined radical/carbocationic models have been proposed to explain the cyclooxygenase part of this reaction. We have used density functional theory calculations to study the mechanism of the formation of the cyclooxygenated product PGG₂. We found an activation free energy for the initial hydrogen abstraction by the tyrosine radical of 15.6 kcal/mol, and of 14.5 kcal/mol for peroxo bridge formation, in remarkable agreement with the experimental value of 15.0 kcal/mol. Subsequent steps of the radical-based mechanism were found to happen with smaller barriers. A combined radical/carbocation mechanism proceeding through a sigmatropic hydrogen shift was ruled out, owing to its much larger activation free energy of 36.5 kcal/mol. Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00214-003-0476-9. Electronic Supplementary MaterialSupplementary material is available in the online version of this article at Electronic Supplementary Material: Supplementary material is available in the online version of this article at     

Synthesis of nonproteinogenic amino acids to probe lantibiotic biosynthesis

The synthesis of six nonproteinogenic amino acids appropriately protected for Fmoc-based solid-phase peptide synthesis is described. These amino acids are (2S,3R)-vinylthreonine, (2S)-(E)-2-amino-5-fluoro-pent-3-enoic acid (fluoroallylglycine), (S)-beta(2)-homoserine, (S) and (R)-beta(3)-homocysteine, and (2R,3R)-methylcysteine. Once incorporated into peptides, these compounds were envisioned to serve as alternative substrates for the lantibiotic synthases that dehydrate serine and threonine residues in their peptide substrates and catalyze the subsequent intramolecular Michael-type addition of cysteines to the dehydroamino acids.     

Selective incorporation of isotopically labeled amino acids for identification of intact proteins on a proteome-wide level

The post-genomic era and increased demands for broad proteome measurements have greatly increased the needs for protein identification. We describe a strategy that uses accurate mass measurements and partial amino acid content information to unambiguously identify intact proteins, and show its initial application to the proteomes of Escherichia coli and Saccharomyces cerevisiae. Proteins were extracted from the organisms grown in minimal medium or minimal medium to which isotopically labeled leucine (Leu-D(10)) had been added. The two protein extracts were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled residue has no effect on the CIEF separation of proteins, and both isotopically labeled and unlabeled versions of specific proteins are observed within the same mass spectrum. The difference in the mass of the unlabeled and labeled proteins is used to determine the number of Leu residues present in a particular protein. Proteins can then often be unambiguously identified based on their accurately determined molecular mass and the additional constraint provided by number of Leu residues. The identities of proteins were further confirmed by repeating CIEF/FTICR measurements with samples that contain other isotopically labeled amino acid residues (e.g. His, Arg, Ile, Phe, Lys). A theoretical study of the amino acid composition (for a difference in the amino acid sequence) showed the constraints needed in order to identify the protein unambiguously. Additionally, the mass differences between the predicted and the experimental accurate mass measurement provide insights into the nature of simple post-translational modifications.     

Synthesis of two isotopically labeled versions of NEDD8-activating enzyme (NAE) inhibitor

The synthesis of two isotopically labeled versions of an investigational NAE inhibitor (MLN4924) is reported. ¹⁴C-MLN4924 was synthesized from ¹⁴C-thiourea in eight steps. D₈-MLN4924 was prepared from D₉-3-phenylpropanoic acid in seven steps.     

Fourier-transform infrared studies of the thermal degradation of isotopically labeled polyacrylonitriles

Poly(d₃-acrylonitrile), poly(1-¹³C-acrylonitrile), poly(¹⁵N-acrylonitrile), and ¹⁸O₂ have been used to assign the infrared spectra of the thermal degradation products of polyacrylonitrile. Isotopic data and elemental analyses show that the degradation products can be made free of oxygen. The infrared data suggest that the reaction proceeds sequentially from imines to fused pyridine-dihydropyridine moieties.     

Study of mechanism of prins reaction employing the method of labeled molecules

Conclusions The use of radiocarbon to study the Prins reaction in aqueous medium disclosed that it proceeds consecutively with the formation of a 1,3-diol as the intermediate product.Deceased.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 8, pp. 1870–1872, August, 1973.     

Synthesis of isotopically labeled puromycin derivatives for kinetic isotope effect analysis of ribosome catalyzed peptide bond formation

The mechanism by which the ribosome catalyze peptide bond formation remains controversial. Here we describe the synthesis of dinucleotides that can be used in kinetic isotope effect experiments to assess the transition state of ribosome catalyzed peptide bond formation. These substrates are the isotopically labeled dinucleotide cytidylyl-(3′-5′)-3′-amino-3′-deoxy-3′-l-phenylalanyl-N⁶,N⁶-dimethyladenosine (Cm⁶A_NPhe-NH₂) and cytidylyl-(3′-5′)-3′-amino-3′-deoxy-3′-(l-2-hydroxy-3-phenylpropionyl)-N⁶,N⁶-dimethyladenosine (Cm⁶A_NPhe-OH). These substrates are active in peptide bond formation and can be used to measure kinetic isotope effects in ribosome catalyzed protein synthesis.     

Probing the reaction mechanism of aristolochene synthase with 12,13-difluorofarnesyl diphosphate

12,13-Difluorofarnesyl diphosphate, prepared using Suzuki-Miyaura chemistry, is a potent inhibitor of aristolochene synthase (AS), indicating that the initial cyclisation during AS catalysis generates germacryl cation in a concerted reaction.     

Synthesis of fluorescently labeled UDP-GlcNAc analogues and their evaluation as chitin synthase substrates

[structure: see text] Chitin synthase (CS) polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc), a key component of fungal cell wall biosynthesis. Little is known about the substrate specificity of chitin synthase or the scope of substrate modification the enzyme will tolerate. Following a previous report suggesting that 6-O-dansyl GlcNAc is biosynthetically incorporated into chitin, we became interested in developing an assay for CS activity based on incorporation of a fluorescent substrate. We describe the synthesis of two fluorescent UDP-GlcNAc analogues and their evaluation as chitin synthase substrates.     

A quantum chemical study of the reaction mechanism of acetyl-coenzyme a synthase

Recent experimental and theoretical studies have focused on the mechanism of the A-cluster active site of acetyl-CoA synthase that produces acetyl-CoA from a methyl group, carbon monoxide, and CoA. Several proposals have been made concerning the redox states of the (Ni-Ni) bimetallic center and the iron-sulfur cluster connected to one of the metals. Using hybrid density functional theory, we have investigated putative intermediate states from the catalytic cycle. Among our conclusions are the following: (i) the zerovalent state proposed for the proximal metal is unlikely if the charge on the iron-sulfur cluster is +2; (ii) a mononuclear mechanism in which both CO and CH(3) bind the proximal nickel is favored over the binuclear mechanism in which CO and CH(3) bind the proximal and distal nickel ions, respectively; (iii) the formation of a disulfide bond in the active site could provide the two electrons necessary for the reaction but only if methylation occurs simultaneously; and (iv) the crystallographic closed form of the active site needs to open to accommodate ligands in the equatorial site.     

Synthesis of chiral chromium tricarbonyl labeled thymine PNA monomers via the Ugi reaction

[reaction: see text] Ugi condensation was used to synthesize the first examples of chiral racemic Ar.Cr(CO)(3) labeled peptide nucleic acid (PNA) monomers bearing the organometallic moiety linked to the alpha-carbon of the glycine unit.     

Oxalate/hydrogen peroxide chemiluminescence reaction. A 19F NMR probe of the reaction mechanism

The mechanism of the oxalate/hydrogen peroxide chemiluminescence reaction has been examined by magnetic resonance techniques. Investigation of the reactive intermediates involved in chemiluminescence was carried out with bis(2,6-difluorophenyl)oxalate (DFPO) using 19F NMR to probe its reactions with aqueous hydrogen peroxide. Formation and reactions of the intermediate hydroperoxy oxalate ester B along with the formation of the half ester product C and difluorophenol D were monitored by 19F NMR. When the reaction of DFPO and aqueous hydrogen peroxide was carried out in the presence of dansylphenylalanine, a typical fluorescent analyte, the intensity of the resonance due to the intermediate B was diminished in direct proportion to the concentration of the analyte. Comparison of the time/intensity profile of the chemiluminescence emission with that of the 19F NMR transient suggests that the hydroperoxy oxalate ester B is the likely 'reactive' intermediate, capable of participating in a chemically initiated electron exchange luminescence mechanism.