Measuring intracellular calcium fluxes in high throughput mode

The measurement of intracellular calcium fluxes in real time is widely applied within the pharmaceutical industry to measure the activation of G-protein coupled receptors (GPCRhyp;s), either for pharmacological characterisation or to screen for new surrogate ligands. Initially restricted to G(q) coupled GPCRs, the introduction of promiscuous and chimeric G-proteins has further widened the application of these assays. The development of new calcium sensitive dyes and assays has provided sensitive, homogeneous assays which can be readily applied to high throughput screening (HTS). In this paper we describe the full automation of this assay type using a fluorometric imaging plate reader (FLIPR ) integrated into a Beckman/Sagian system to establish a simple robotic system that is well suited for the current medium throughput screening in this area of lead discovery. Using a recently completed HTS we discuss important determinants for FLIPR based screening, highlight some limitations of the current approach, and look at the requirements for future automated systems capable of keeping up with expanding compound files.     

Transannular reactions of two parallel 1,3-butadiynes: syntheses,structures, and reactions of 1-azacyclotetradeca-3,5,10,12-tetrayne derivatives

The synthesis of 1-alkyl and 1-aryl-1-azacyclotetradeca-3,5,10,12-tetraynes was achieved in a stepwise approach. The key intermediate was 1,13-dibromotrideca-2,4,9,11-tetrayne (18). Reaction with methyl- (19 a), ethyl- (19 b), isopropyl- (19 c), n-butyl- (19 d), and tert-butylamine (19 e) as well as aniline (19 f) and p-methoxyaniline (19 g) gave the corresponding 14-membered tetraynes 20 a-20 g. The ring inversion process of 20 b was studied by variable temperature (1)H NMR spectroscopy. From these measurements a value of 10.6 kcal mol(-1) was calculated for DeltaG(not equal). X-ray investigations on single crystals of 20 b, 20 c, and 20 f revealed the axial position for the substituent at each nitrogen atom. For 20 b we encountered the chair conformation, for 20 c both chair and boat conformations, and for 20 f the boat conformation in the solid state. The reaction of 20 c with concentrated HCl in ethanol yielded 2,10-dichloro-6-isopropyl-6-azatricyclo[9.3.0.0(4,8)]tetradeca-1(11),2,4(8),9-tetraene (25 c). Compound 25 c was oxidized by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to 27 c. The structure of the latter was confirmed by X-ray investigations. The reaction of 20 c in aqueous HCl lead to the formation of 10-chloro-2-isopropyl-1,3,4,6,7,8-hexahydro-2H-benzo[g]isoquinolin-9-one (37 c). The structure of 37 c was verified by X-ray studies on single crystals.     

Mechanism of silver-promoted ligand metathesis in square-planar complexes of d(8) Ions. Kinetics of formation and molecular structures of a trinuclear intermediate [(Me)(N-N)Pt(mu-Cl)Ag(mu-Cl)Pt(N-N)(Me)](+) and its dinuclear evolution product [(Me)(N-N)Pt(mu-Cl)Pt(N-N)(Me)](+) (N-N = ArN=C(Me)C(Me)=NAr,Ar = 2,6-(i-Pr)(2)C(6)H(3))

The silver-assisted ligand metathesis reaction involving a platinum(II) complex of formula [PtClMe(N,N-chelate)] with acetonitrile has been investigated. By using a suitably hindered N,N-chelate, an otherwise hardly detectable trinuclear species has been isolated and characterized through X-ray diffractometry. The trinuclear cation consists of two nearly orthogonal [PtCl(Me)(N,N-chelate)] square-planar units entrapping an Ag(+) cation through the chloride ligands that, acting as bidentate, form a linear AgCl(2) unit with two nonequivalent Ag-Cl bonds. The residual acidity of the silver cation is satisfied by one secondary Ag-Pt interaction [Ag-Pt(1) = 2.82 A] in which the platinum atom acts as a donor. Kinetic studies have demonstrated that the silver assistance operates both through a simple associative step and through a pathway in which the above trinuclear complex is an active intermediate. In a noncoordinating solvent the latter species evolves with AgCl loss and formation of a dinuclear Pt,Pt complex showing a rare single chloride bridge.     

Pilot plant study of the microbial flora in a diesel fuel contaminated soil

This study focused on the impact of a recent soil pollution of diesel fuel on a site and its indigenous microbial flora. A pilot plant (0.5 m3) was set up and filled with a soil (about 700 kg), artificially and uniformly polluted with diesel fuel (7 g/kg). This plant was then chemically and biologically monitored during the whole experiment (about two years). During the monitoring, a morphological change of the microbial colonies was observed. This was probably due to the acclimation phenomena to the pollution. With batch kinetic studies (10 ml) and increasing the selective pressure of the pollutant, it was possible to select and isolate a microbial consortium and a single strain that developed the ability to use different diesel fuel fractions as carbon sources. GC-MS analytical techniques were used. Results showed that different fractions were degraded at different times. In the batch system, in 7 days, the microbial consortium degraded some aromatic hydrocarbons. The isolate strain, in 20 days, degraded linear hydrocarbons. After a two years acclimation, it was possible to obtain, from a pilot plant, a microbial consortium and a strain able to degrade diesel fuel, for a future bioremediation in situ process.     

Synthesis of 11C-amides using [11C]carbon monoxide and in situ activated amines by palladium-mediated carboxaminations

[11C]Carbon monoxide at low concentrations, aryl halides and amines were used in the palladium-mediated synthesis of twenty 11C-amides. In the study several approaches to improve the radiochemical yield were explored. Eight of the selected amides were prepared by in situ activation of the amines using lithium bis(trimethylsilyl)amide and the radiochemical yields of these reactions were improved compared to utilising a previous reported method. In the synthesis of 1-[carbonyl-11C]benzoyl-3-methyl-1H-indole (11) from 3-methyl-1H-indole (25), the corresponding organotin-amine was prepared prior to the acylation reaction. In a typical experiment, N-(4-hydroxyphenyl)[carbonyl-11C]acetamide (5) was prepared in 15% radiochemical yield using 4-aminophenol (20) but the yield increased to 63% when the amine was activated by lithium bis(trimethylsilyl)amide.     

Trinuclear heterobimetallic complexes with binucleating dithioxamides: stereoselective synthesis and solution behavior involving Pd-N bond rupture

Bischelate platinum(II) complexes of the type [Pt(H-R(2)-N(2)C(2)S(2))(2)] (H-R(2)-N(2)C(2)S(2)(-) = dialkyl-dithioxamidate) are ditopic receptors which, after coordination of the first Pd(eta(3)-allyl)(+) moiety, induce the orientation of the second palladium-allyl fragment. Thus, a series of trimetallic complexes of formula bis-[(eta(3)-allyl)-palladium(II)](mu-bis-dialkyl-dithioxamidate-platinum(II) kappa-S,S-kappa-S',S'-Pt-kappa-N,N-Pd-kappa-N',N'-Pd') has been prepared in which the allyl fragments are oriented toward the same side of the molecular plane. We have also prepared the trimetallic complex using a dithioxamide obtained from the racemic phenylethylamine. Only two isomers were produced in equimolar ratio: the racemate that has four homochiral alkyl substituents and the mesoform containing the meso-dithioxamide that has homochiral substituents on the same side of molecular plane. Under the effect of the temperature, the trimetallic Pd-Pt-Pd complexes undergo rapid allyl isomerization; the mechanism of the isomerization, which is similar to that found by us in an analogue Pt-Pd bimetallic complex, is discussed. The crystal and molecular structure of bis-[(eta(3)-allyl)-palladium(II)](mu-bis-[S]-phenylethyl-dithioxamidate-platinum(II) kappa-S,S-kappa-S',S'-Pt-kappa-N,N-Pd-kappa-N',N'-Pd') has been reported.     

GC-MS in space research: decoding complex isothermal chromatograms recovered from space missions

An analytical procedure is described to study GC-MS isothermal chromatograms simulating those recovered from space missions: in fact GC plays a predominant role in space missions devoted to characterizing the chemical composition of extra-terrestrial atmospheres. SIM (selected ion monitoring) detection was used for monitoring selected chemical classes: a simplified chromatogram can be obtained giving information on the chemical composition of the complex mixture. Since only isothermal GC chromatograms are allowed by flight constraints, a time axis transformation is required to make them homogeneous: i.e., constant retention increments for CH2 additions in terms of a homologous series. The order in the linearized chromatogram can be simply singled out with a chemometric approach based on the study of the Autocovariance Function (ACVF) computed on the digitized chromatogram: the plot of the experimental autocorrelation function (EACF) shows well-shaped peaks if constant interdistances are repeated in different regions of the chromatogram. The method was applied to standard mixtures representative of planetary atmospheres--hydrocarbons, nitriles and oxygenated compounds with between 3 and 12 carbon atoms--analyzed in flight simulating conditions. The coupling of the selectivity of SIM detection with the interpretation power of the EACF procedure proves to be a powerful tool for interpreting data recovered from space missions: the chemical composition of the mixture can be identified by handling the raw SIM chromatograms.     

Kinetic and mechanistic studies on the reaction of nitric oxide with a water-soluble octa-anionic iron(III) porphyrin complex

The polyanionic water-soluble and non-mu-oxo-dimer-forming iron porphyrin iron(III) 5(4),10(4),15(4),20(4)-tetra-tert-butyl-5(2),5(6),15(2),15(6)-tetrakis[2,2-bis(carboxylato)ethyl]-5,10,15,20-tetraphenylporphyrin, (P(8-))Fe(III) (1), was synthesized as an octasodium salt by applying well-established porphyrin and organic chemistry procedures to bromomethylated precursor porphyrins and characterized by standard techniques such as UV-vis and (1)H NMR spectroscopy. A single pK(a1) value of 9.26 was determined for the deprotonation of coordinated water in (P(8-))Fe(III)(H(2)O)(2) (1-H(2)()O) present in aqueous solution at pH <9. The porphyrin complex reversibly binds NO in aqueous solution to give the mononitrosyl adduct, (P(8-))Fe(II)(NO(+))(L), where L = H(2)O or OH(-). The kinetics of the binding and release of NO was studied as a function of pH, temperature, and pressure by stopped-flow and laser flash photolysis techniques. The diaqua-ligated form of the porphyrin complex binds and releases NO according to a dissociative interchange mechanism based on the positive values of the activation parameters DeltaS() and DeltaV() for the "on" and "off" reactions. The rate constant k(on) = 6.2 x 10(4) M(-1) s(-1) (24 degrees C), determined for NO binding to the monohydroxo-ligated (P(8-))Fe(III)(OH) (1-OH) present in solution at pH >9, is markedly lower than the corresponding value measured for 1-H(2)O at lower pH (k(on) = 8.2 x 10(5) M(-1) s(-1), 24 degrees C, pH 7). The observed decrease in the reactivity is contradictory to that expected for the diaqua- and monohydroxo-ligated forms of the iron(III) complex and is accounted for in terms of a mechanistic changeover observed for 1-H(2)O and 1-OH in their reactions with NO. The mechanistic interpretation offered is further substantiated by the results of water-exchange studies performed on the polyanionic porphyrin complex as a function of pH, temperature, and pressure.     

Ultrafast energy-electron transfer cascade in a multichromophoric light-harvesting molecular square

A molecular square with dimensions of about 4 nm, incorporating sixteen pyrene chromophores attached to four ditopic bay-functionalized perylene bisimide chromophores, has been synthesized by coordination to four Pt(II) phosphine corner units and fully characterized via NMR spectroscopy and ESI-FTICR mass spectrometry. Steady-state and time-resolved emission as well as femtosecond transient absorption studies reveal the presence of a highly efficient (>90%) and fast photoinduced energy transfer (k(en) approximately equal to 5.0 x 10(9) s(-1)) from the pyrene to the perylene bisimide chromophores and a very fast and efficient electron transfer (>94%, k(et) approximately equal to 5 x 10(11) up to 43 x 10(11) s(-1)). Spectrotemporal parametrization indicates upper excited-state electron-transfer processes, various energy and electron-transfer pathways, and chromophoric heterogeneity. Temperature-dependent time-resolved emission spectroscopy has shown that the acceptor emission lifetime increases with decreasing temperature from which an electron-transfer barrier is obtained. The extremely fast electron-transfer processes (substantially faster and more efficient than in the free ligand) that are normally only observed in solid materials, together with the closely packed structure of 20 chromophoric units, indicate that we can consider the molecular square as a monodisperse nanoaggregate: a molecularly defined ensemble of chromophores that partly behaves like a solid material.     

Why copper is intrinsically more selective than silver in alkene epoxidation: ethylene oxidation on Cu(111) versus Ag(111)

The heterogeneously catalyzed epoxidation of alkenes is experimentally challenging, theoretically interesting, and technologically important. Although large-scale ethylene epoxidation is universally carried out with Ag catalysts, recent laboratory studies on single crystal surfaces show that Cu is intrinsically much more selective than Ag in the epoxidation of a variety of terminal alkenes. The reasons for this striking difference between Ag and Cu have been investigated by means of density functional theory. It is found that the fundamental cause is the inversion in the ordering of activation barriers for the competing pathways to epoxide formation versus acetaldehyde formation (the latter being the first step on the route to combustion). On Cu, epoxide formation is less activated than aldehyde formation; the opposite is true on Ag. This behavior is associated with a late transition state to epoxidation on Cu (i.e., product-like) compared to an early (reactant-like) transition state to epoxidation on Ag.