A rapid fluorometric assay for the determination of serum phospholipase A2 activity

Phospholipase A₂ (PLA₂) is an enzyme that cleaves fatty acids from the sn-2 position of membrane phospholipids. Intracellular PLA₂ has been shown to be important for the generation of arachidonic acid, which leads to the synthesis of a wide range of eicosanoid paracrine hormones. Furthermore, secretory PLA₂ in the serum has been reported to exhibit important immune activities. In this study, BODIPY™ FL C₁₆ was used as a fluorescent probe to measure PLA₂ activity in the serum of the American alligator. The activity measured was dependent on the amounts of serum and labeled bacteria incubated. The phospholipase activity was also time-dependent, with near peak activities observed within 20-30 min. In addition, the observed activity was titrated away by increasing concentrations of unlabeled bacteria, indicating that the activity observed was specifically due to the presence of PLA₂. The assay provides rapid results and requires only small amounts of serum.     

Quantum dot–NBD–liposome luminescent probes for monitoring phospholipase A2 activity

In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot–fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A_2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C₆-HPC (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A_2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A₂ as low as 0.0075 U mL^?1 and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A₂ inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).     

Involvement of the γ Isoform of cPLA2 in the Biosynthesis of Bioactive N-Acylethanolamines

Arachidonylethanolamide (anandamide) acts as an endogenous ligand of cannabinoid receptors, while other N-acylethanolamines (NAEs), such as palmitylethanolamide and oleylethanolamide, show analgesic, anti-inflammatory, and appetite-suppressing effects through other receptors. In mammalian tissues, NAEs, including anandamide, are produced from glycerophospholipid via N-acyl-phosphatidylethanolamine (NAPE). The ɛ isoform of cytosolic phospholipase A₂ (cPLA₂) functions as an N-acyltransferase to form NAPE. Since the cPLA₂ family consists of six isoforms (α, β, γ, δ, ɛ, and ζ), the present study investigated a possible involvement of isoforms other than ɛ in the NAE biosynthesis. Firstly, when the cells overexpressing one of the cPLA₂ isoforms were labeled with [¹⁴C]ethanolamine, the increase in the production of [¹⁴C]NAPE was observed only with the ɛ-expressing cells. Secondly, when the cells co-expressing ɛ and one of the other isoforms were analyzed, the increase in [¹⁴C]N-acyl-lysophosphatidylethanolamine (lysoNAPE) and [¹⁴C]NAE was seen with the combination of ɛ and γ isoforms. Furthermore, the purified cPLA₂γ hydrolyzed not only NAPE to lysoNAPE, but also lysoNAPE to glycerophospho-N-acylethanolamine (GP-NAE). Thus, the produced GP-NAE was further hydrolyzed to NAE by glycerophosphodiesterase 1. These results suggested that cPLA₂γ is involved in the biosynthesis of NAE by its phospholipase A₁/A₂ and lysophospholipase activities.     

Zeta potential and surface free energy changes of solid-supported phospholipid (DPPC) layers caused by the enzyme phospholipase A2 (PLA2)

Stability and wetting properties changes of systems formed of phospholipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) layers covering silica particles or glass slides due to the phospholipase A₂ (PLA₂) action were determined by zeta potential measurements and the surface free energy evaluation, respectively. The comparison of the zeta potential and surface free energy, which was evaluated from advancing and receding contact angles via applying models of interfacial interactions, i.e. van Oss et al. (LWAB) and contact angle hysteresis (CAH), was found to be helpful for better understanding the mechanism of PLA₂ action on the lipid layers, what is discussed in the paper.     

Singlet-triplet energy separations in divalent five-membered cyclic conjugated C5H3X, C4H3SiX, C4H3GeX, C4H3SnX, and C4H3PbX (X = H, F, Cl, and Br)

Singlet-triplet energy gaps in cyclopenta-2,4-dienylidene, as well as its 2- or 3-halogenated derivatives, are compared and contrasted with their sila, germa, stana, and plumba analogues; at HF/6-31G* and B3LYP/ 6-311++G(3df, 2p) levels of theory. Energy gaps (ΔG_t-s), between triplet (t) and singlet (s) states, appear linearly proportional to: (i) the size of the group 14 divalent element (M = C, Si, Ge, Sn and Pb), (ii) the angle ∠C-M-C, and (iii) the ΔG_(LUMO-HOMO) of the singlet state involved. The magnitude of ΔG_t-s, for each 2- and/or 3-substituted species studied, increases with an order of: carbenes < silylenes < germylenes < stanylenes < plumbylenes. This order reverses for the barriers of the ring puckering. The puckering occurs with more ease for every singlet, compared to its corresponding triplet form.Regardless of the group 14 element (M) employed, every 3-halo-substituted species is more stable than the corresponding 2-halo-substituted isomer. For M = Pb, Sn and/or Ge; 3-halo-substituted species have higher ΔG_t-s than their corresponding 2-halo-substituted analogues. For M = Si, similar ΔG_t-s are found for 2- and 3-halogenated isomers. For M = C, 3-halo-substituted species have lower ΔG_t-s than their corresponding 2-halo-substituted analogues.Every cyclic singlet has a larger ∠C-M-C angle, than its corresponding cyclic triplet state, except for 3-halosilacyclopenta-2,4-dienylidenes where triplet has a larger ∠C-M-C angle than its corresponding singlet state.     

Effect of S–Se Bioisosteric Exchange on Affinity and Intrinsic Efficacy of Novel N-acylhydrazone Derivatives at the Adenosine A2A Receptor

In this work, we evaluated the conformational effect promoted by the isosteric exchange of sulfur by selenium in the heteroaromatic ring of new N-acylhydrazone (NAH) derivatives (3–8, 13, 14), analogues of the cardioactive compounds LASSBio-294 (1) and LASSBio-785 (2). NMR spectra analysis demonstrated a chemical shift variation of the iminic Csp² of NAH S/Se-isosters, suggesting a stronger intramolecular chalcogen interaction for Se-derivatives. To investigate the pharmacological profile of these compounds at the adenosine A_2A receptor (A_2AR), we performed a previously validated functional binding assay. As expected for bioisosteres, the isosteric-S/Se replacement affected neither the affinity nor the intrinsic efficacy of our NAH derivatives (1–8). However, the N-methylated compounds (2, 6–8) presented a weak partial agonist profile at A_2AR, contrary to the non-methylated counterparts (1, 3–5), which appeared as weak inverse agonists. Additionally, retroisosterism between aromatic rings of NAH on S/Se-isosters mimicked the effect of the N-methylation on intrinsic efficacy at A_2AR, while meta-substitution in the phenyl ring of the acyl moiety did not. This study showed that the conformational effect of NAH-N-methylation and aromatic rings retroisosterism changed the intrinsic efficacy on A_2AR, indicating the S/Se-chalcogen effect to drive the conformational behavior of this series of NAH.     

Design and Synthesis of Arylpiperazine Serotonergic/Dopaminergic Ligands with Neuroprotective Properties

Long-chain arylpiperazine scaffold is a versatile template to design central nervous system (CNS) drugs that target serotonin and dopamine receptors. Here we describe the synthesis and biological evaluation of ten new arylpiperazine derivatives designed to obtain an affinity profile at serotonin 5-HT_1A, 5-HT_2A, 5-HT₇ receptor, and dopamine D₂ receptor of prospective drugs to treat the core symptoms of autism spectrum disorder (ASD) or psychosis. Besides the structural features required for affinity at the target receptors, the new compounds incorporated structural fragments with antioxidant properties to counteract oxidative stress connected with ASD and psychosis. All the new compounds showed CNS MultiParameter Optimization score predictive of desirable ADMET properties and cross the blood–brain barrier. We identified compound 12a that combines an affinity profile compatible with antipsychotic activity (5-HT_1A K_i = 41.5 nM, 5-HT_2A K_i = 315 nM, 5-HT₇ K_i = 42.5 nM, D₂ K_i = 300 nM), and compound 9b that has an affinity profile consistent with studies in the context of ASD (5-HT_1A K_i = 23.9 nM, 5-HT_2A K_i = 39.4 nM, 5-HT₇ K_i = 45.0 nM). Both compounds also had antioxidant properties. All compounds showed low in vitro metabolic stability, the only exception being compound 9b, which might be suitable for studies in vivo.     

Linear and convergent approaches to 2-substituted adenosine-5′-N-alkylcarboxamides

Herein we report both linear and convergent pathways for the preparation of 2-alkynyl substituted adenosine-5′-N-ethylcarboxamides via the versatile synthetic intermediate, 2-iodoadenosine-5′-N-ethylcarboxamide (13). The linear approach afforded 13 in an overall yield of 30% from guanosine over eight synthetic steps. The convergent approach was shorter, but proceeded in lower yield (five steps, 20% yield). Both approaches compare favourably with previously reported syntheses of 13, which has been prepared in 15% yield from guanosine over nine steps. 2-Iodoadenosine-5′-N-ethylcarboxamide (13) was subsequently converted to HENECA (2) and PHPNECA (3) to exemplify the utility of this approach for the preparation of potent A_2A adenosine receptor agonists. The linear approach was also amenable to the synthesis of 2-fluoropurine ribosides, which were subsequently elaborated into 2-alkylaminoadenosine-5′-N-ethylcarboxamides. Furthermore, both of these synthetic approaches are readily amenable to the synthesis of adenosine analogues with varied 2-, 6- and 5′-substitution patterns.     

Synthesis,Antitumor and Antibacterial Studies of New Shortened Analogues of (KLAKLAK)2-NH2 and Their Conjugates Containing Unnatural Amino Acids

(1) Background: (KLAKLAK)₂ is a representative of the antimicrobial peptide group which also shows good anticancer properties. (2) Methods: Herein, we report synthesis using SPPS and characterization by HPLC/MS of a series of shortened analogues of (KLAKLAK)₂. They contain single sequence KLAKLAK as C-terminal amides. In addition, substitution of some natural amino acids with unnatural β-Ala and nor-Leu is realized. In addition, these structures are conjugated with second pharmacophore with well proven anticancer properties 1,8-naphthalimide or caffeic acid. Cytotoxicity, antiproliferative effect and antimicrobial activity of newly synthesized structures were studied. (3) Results: The obtained experimental results reveal significant selective index for substances with common chemical structure KLβAKLβAK-NH₂. The antibacterial properties of newly synthesized analogues at two different concentrations 10 μM and 20 μM, were tested against Gram-negative microorganisms Escherichia coli K12 407. Only two of the studied compounds KLAKLAK-NH₂ and the one conjugated with second pharmacophore 1,8-naphthalimide and unnatural amino acid nor-Leu showed moderate activity against tested strains at concentration of 20 μM. (4) Conclusions: The obtained results reveal that the introducing of 1,8-naphthalimideGly- and Caf- increase the cytotoxicity and antiproliferative activity of the peptides but not their selectivity. Only two compounds KLAKLAK-NH₂ and 1,8-naphthalimideGKnLAKnLAK-NH₂ show moderate activity against Escherichia coli K12 at low concentration of 20 μM.     

Structural studies of five layer Aurivillius oxides: A2Bi4Ti5O18 (A=Ca, Sr, Ba and Pb)

The room temperature structures of the five layer Aurivillius phases A₂Bi₄Ti₅O₁₈ (A=Ca, Sr, Ba and Pb) have been refined from powder neutron diffraction data using the Rietveld method. The structures consist of [Bi₂O₂]²⁺ layers interleaved with perovskite-like [A₂Bi₂Ti₅O₁₆]²⁻ blocks. The structures were refined in the orthorhombic space group B2eb (SG. No. 41), Z=4, and the unit cell parameters of the oxides are a=5.4251(2), b=5.4034(1), c=48.486(1); a=5.4650(2), b=5.4625(3), c=48.852(1); a=5.4988(3), b=5.4980(4), c=50.352(1); a=5.4701(2), b=5.4577(2), c=49.643(1) for A=Ca, Sr, Ba and Pb, respectively. The structural features of the compounds were found similar to n=2-4 layers bismuth oxides. The strain caused by mismatch of cell parameter requirements for the [Bi₂O₂]²⁺ layers and perovskite-like [A₂Bi₂Ti₅O₁₆]²⁻ blocks were relieved by tilting of the TiO₆ octahedra. Variable temperature synchrotron X-ray studies for Ca and Pb compounds showed that the orthorhombic structure persisted up to 675 and 475 K, respectively. Raman spectra of the compounds are also presented.     

Fast method for monitoring phospholipase A2 activity by liquid chromatography–electrospray ionization mass spectrometry

A new liquid chromatography–electrospray ionization mass spectrometry (LC–ESI-MS) method for the fast determination of phospholipase A₂ (PLA₂) activity has been developed. For the first time, the method allows the parallel detection of glycerophosphatidylcholine (GroPCho) as PLA₂ substrate as well as of its products fatty acid (FA) and lyso-GroPCho. ESI-MS was carried out in negative ion mode, detecting the FA as [M − H]⁻ ions and the lyso-GroPCho and GroPCho as acetate adducts [M + Ac]⁻. Utilizing a fast gradient on a short C₅-modified silica gel column with 3 μm particles, five GroPChos, five FAs and six lyso-GroPChos could be separated according to their chain length in less than 3 min. A very high average chromatographic efficiency of 41,200 theoretical plates (plate height 0.5 μm) was achieved for the separation of the GroPChos. The method was applied for monitoring the release of arachidonic acid (20:4 FA) and 1-stearoyl-lyso-sn-GroPCho (18:0 GroPCho) from unilamellar vesicles of 1-stearoyl-2-arachidonoyl-sn-GroPCho (18:0/20:4 GroPCho). With a limit of detection of 0.5 pmol (total amount injected on column) for the FAs and lyso-GroPChos and 1.5 pmol for the GroPChos as well as a linear range of 1.5 decades, the method has proven to be suitable for the monitoring of different secretory PLA₂ (sPLA₂) conversions. Furthermore, it was applied to screen a small library of PLA₂ inhibitors for their activity towards sPLA₂ type V and snake venom of Bothrops moojeni. In both cases, active samples could be directly identified. With its short analysis time, its high chromatographic efficiency and the parallel detection of substrate and all products, the developed LC–ESI-MS method is well suited for the analysis of PLA₂ activity.     

Magnetic and charge transport properties of the Na-based Os oxide pyrochlore

The Na-based osmium oxide pyrochlore was synthesized for the first time by an ion-exchange method using KOs₂O₆ as a host. The composition was identified as Na_1.4Os₂O₆·H₂O by electron probe micro-analysis, thermogravimetric analysis, and structural analysis using synchrotron X-ray diffraction. Na_1.4Os₂O₆·H₂O crystallizes in a regular pyrochlore structure with some defects (space group: Fd-3m, a=10.16851(1) Å). Electrical resistivity, heat capacity, and magnetization measurements clearly showed absence of superconductivity down to 2 K, being in large contrast to what was found for the β-type pyrochlore superconductor AOs₂O₆ (A=Cs, Rb, and K). The Sommerfeld coefficient is 22 mJ K⁻² mol⁻¹, being the smallest among AOs₂O₆. A magnetic anomaly at ∼57 K and associated magneto-resistance (+3.7% at 2 K in 70 kOe) were found.     

A structure, conductivity and dielectric properties investigation of A3CoNb2O9 (A=Ca, Sr, Ba) triple perovskites

The room temperature structures as well as the temperature-dependent conductivity and dielectric properties of the A₃CoNb₂O₉ (A=Ca²⁺, Sr²⁺ and Ba²⁺) triple perovskites have been carefully investigated. A constrained modulation wave approach to Rietveld structure refinement is used to determine their room temperature crystal structures. Correlations between these crystal structures and their physical properties are found. All three compounds undergo insulator to semiconductor phase transitions as a function of increasing temperature. The hexagonal Ba₃CoNb₂O₉ compound acts as an insulator at room temperature, while the monoclinic Ca₃CoNb₂O₉ compound is already a semiconductor at room temperature. The measured dielectric frequency characteristics of the A=Ba compound are excellent.     

Antibacterial Properties of Intestinal Phospholipase A2 from the Common Stingray Dasyatis pastinaca

Stingray phospholipase A₂ group IIA (SPLA₂-IIA) was recently isolated and purified to homogeneity from the intestine of the common stingray Dasyatis pastinaca, suggesting that this enzyme plays an important role in systemic bactericidal defense. The present study showed that SPLA₂-IIA was highly bactericidal against Gram-positive bacteria with inhibition zones and minimal inhibitory concentration values in the range of 13–25 mm and 2–8 μg/ml, respectively, whereas Gram-negative bacteria exhibited a much higher resistance. The bactericidal efficiency of SPLA₂-IIA was shown to be unaffected by high protein and salt concentrations, but dependent upon the presence of calcium ions, and then correlated to the hydrolytic activity of membrane phospholipids. Importantly, we showed that stingray phospholipase A₂ group IIA presents no cytotoxicity after its incubation with MDA-MB-231 cells. SPLA₂-IIA may be considered as a future therapeutic agent against bacterial infections.     

A new one-pot synthesis of Gb3 and isoGb3 trisaccharide analogues

Gb₃ and isoGb₃ are both biologically important oligosaccharides. A new efficient synthesis of Gb₃ and isoGb₃ trisaccharide analogues has been achieved by one-pot sequential glycosylation strategy starting from simple monosaccharide building blocks promoted by N-(phenylthio)-?-caprolactam at room temperature.     

Crystal chemistry and crystallography of the Aurivillius phase Bi5AgNb4O18

Bi₅AgNb₄O₁₈ is a new phase, which was discovered during the phase equilibrium study of the Bi₂O₃-Ag₂O-Nb₂O₅ system. Bi₅AgNb₄O₁₈ was prepared at 750°C and is stable in air up to its melting temperature of 1160.1±5.0°C (standard error of estimate). Results of a Rietveld refinement using neutron powder diffraction confirmed that Bi₅AgNb₄O₁₈ is isostructural with Bi₃TiNbO₉, Bi₅NaNb₄O₁₈, and Bi₅KNb₄O₁₈. The structure was refined in the orthorhombic space group A2₁am, Z=2, and the lattice parameters are a=5.4915(2) Å, b=5.4752(2) Å, c=24.9282(8) Å, and V=749.52(4) Å³. The structure can be described as the m=2 member of the Aurivillius family, (Bi₂O₂)²⁺ (A_m−1B_mO_3m+1)²⁻ (where A=Bi and B=Ag, Nb), which is characterized by perovskite-like (A_m−1B_mO_3m+1)²⁻ slabs regularly interleaved with (Bi₂O₂)²⁺ layers. The octahedral [NbO₆] units are distorted with Nb-O distances ranging from 1.856(4) to 2.161(2) Å and the O-Nb-O angles ranging from 82.6(3)° to 98.5(3)°. These octahedra are tilted about the a- and c-axis by about 10.3° and 12.4°, respectively. Ag was found to substitute exclusively into the Bi-site that is located in the layer between the two distorted [NbO₆] units. Although the Ag substitutes into the Bi-site with the Bi:Ag ratio of 1:1, the existence of a superlattice was not detected using electron diffraction. A comparison of (Bi₂O₂)²⁺(A_m−1Nb_mO_3m+1)²⁻ structures (where A=Ag, Na, and K) revealed a relation between the pervoskite tolerance factor, t, and structural distortion. The reference pattern for Bi₅AgNb₄O₁₈ has been submitted to the International Centre for Diffraction Data (ICDD) for inclusion in the Powder Diffraction File.     

Total synthesis of (+)-blastmycinone and formal synthesis of (+)-antimycin A3b

The formal synthesis of (+)-antimycin A_3b and the total synthesis of (+)-blastmycinone were achieved using, as a key step, a method developed by us for the synthesis of 2-methyl-1,3-diols via Ti(III)-mediated diastereo- and regioselective opening of trisubstituted 2,3-epoxy alcohols, to carry out the stereoselective construction of the hydroxy-acid segment. An interesting intramolecular radical translocation took place during the epoxide opening process transforming its vicinal PMB-ether in situ, into an ‘1,2-O-(p-methoxy)benzylidene’ ring.     

Synthesis of fluorinated analogues of the neurosteroid GABAA receptor antagonist, 17-PA

The steroid 17-PA is a GABA_A receptor antagonist which is finding use as a tool in evaluating agonistic/antagonistic activity at GABA_A receptors. Compounds with improved efficacy over 17-PA would be are advantageous for such studies. Accordingly a series of novel analogues of the neurosteroid 17-PA have been prepared and a convenient two-step route is presented which is amenable to the synthesis of analogues with electron-donating para-aromatic substituents including fluorine. However, for the meta-fluoro analogue then the original four-step route to 17-PA remains more efficient overall. The paper describes these syntheses and discusses the electronic factors which influence this synthetic chemistry.     

Phase transitions in A-site substituted perovskite compounds: The (Ca1-2xNaxLax)TiO3 (0?x?0.5) solid solution

The (Ca_1-2xNa_xLa_x)TiO₃ (0?x?0.5) A-site substituted perovskite compounds have been synthesized and characterized by XRD and Raman spectroscopy at room temperature. The XRD powder diffraction study suggests that the end-member Na_1/2La_1/2TiO₃ crystallizes in the tetragonal space group I4/mcm. The phase transition from Pbnm to I4/mcm is located between x=0.34 and 0.39 and is driven by the variation of ionic radii at the A-site. The observed Raman modes are in agreement with group theory analysis, and the relationships between the behavior of structural parameters (e.g. Ti-O-Ti bond angle), indicated by long-range order, and the corresponding Raman frequency shifts and intensity evolution, indicated by short-range order, are established and discussed in terms of the radius effect and the mass effect.