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.     

Synthesis of crystalline skutterudite superlattices using the modulated elemental reactant method

A series of samples ((AB)(x)(CD)(y))(z) were prepared containing both short repeat units (AB and CD) and long repeat units ((AB)(x)(CD)(y)), where the short repeat units were designed to have the composition appropriate to form square M(4)Sb(12) skutterudites (M = Fe, Co, or Ir; square = vacancy, La, or Y). X-ray diffraction and reflectivity were used to follow the evolution of the films from amorphous, layered materials to crystalline skutterudite superlattices as a function of annealing temperature and time. In all cases, the short repeat units interdiffused and crystallized the expected skutterudite, while the long repeat period persisted after annealing. The skutterudites crystallize with random crystallographic orientation with respect to the substrate. The observed splitting of the peaks in the high-angle diffraction data from the IrSb(3)/CoSb(3) sample indicates the formation of a novel superlattice structure with each grain having a random crystallographic orientation of the skutterudite lattice with respect to the superlattice direction.     

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.     

The mechanism of the Fischer-Hepp rearrangement of aromatic N-nitroso-amines

Rate equations have been deduced for two possible mechanisms for the Fischer-Hepp rearrangement of aromatic N-nitroso-amines in acid solution: (a) for the commonly assumed intermolecular process involving de-nitrosation to the secondary amine and a free nitrosating agent, followed by a direct C-nitrosation of the secondary amine by this nitrosating agent, and (b) for a mechanism whereby rearrangement and de-nitrosation occur concurrently, by two separate reactions of the protonated N-nitroso-amine. All the experimental evidence supports mechanism (b), whilst most of it is incompatible with (a). Particularly diagnostic of the mechanism are (1) the observed rearrangement: de-nitrosation product ratios under certain limiting conditions, (2) the question of halide ion catalysis, and (3) the rate form found under the limiting condition of a large excess of added secondary amine.     

Sulfonated polypropylene ionomers

Polypropylene ionomers have been prepared by sulfonation of copolymers of propene and 7 methyl, 1-6 octadiene, followed by neutralization to cesium salts. Both WAXS and SAXS were used to study the morphology of the samples, while their thermal properties were studied by DSC and their mechanical properties by DMTA. The sulfonation process is shown to cause a further drop in crystallinity in addition to the effect of comonomer incorporation. Ion clustering is observed when the extent of sulfonation is high enough, the limit being dependent on the copolymer composition. The ion pairs which are not incorporated into the cluster cause a small-angle upturn in the WAXS pattern. The mechanical properties are strongly affected by the drop in crystallinity, but may be partly recovered due to ion clustering. No disruption of the ion clusters is observed before thermal decomposition of the polymer.     

Preparation and characterization of some cellulose graft copolymers. Part IV. Some properties of isolated cellulose acetate–styrene graft copolymers

A series of purified graft copolymers of cellulose acetate and polystyrene, which were prepared and characterized during the course of an earlier investigation, have been used for some initial property studies. Grafts with high molecular weight backbones and low molecular weight side chains and vice versa, and with both side chains and backbones of high and low molecular weights, were prepared. The graft polymers were found to be insoluble in most solvents but soluble in dimethylformamide and pyridine and in mixtures of solvents for each component. Films cast from mixtures of the grafts and homopolymers showed that the grafts were compatible with the homopolymer of the component with a higher molecular weight. A graft copolymer with high molecular weight backbone and side chains was found to be compatible with both homopolymers separately and with a mixture of both across a wide range of compositions. A blend of the high and low molecular weight grafts was also found to be highly compatible with both homopolymers. The permeability, diffusion, and sorption of gases and water vapor in the grafts and in the corresponding homopolymers was also studied. In the case of water vapor, the sorption in cellulose acetate was much higher than in polystyrene whereas the diffusion was much lower. It was found that the graft polymers showed both diffusion and solution behavior closer to that of cellulose acetate, whereas the permeability constants were more intermediate between those of the two homopolymers. The gas permeability and diffusion constants were found to be intermediate between the values obtained with the two homopolymers.     

N,N-difluorotris(tert-butyl)silylamine-the first organosilyl difluoroamine. Synthesis and computational studies

The synthesis and characterization of the first stable trialkyl(difluoroamino)silane, R3SiNF2, as well as of R3SiNHF and R3SiN(CH3)F in moderate yields are reported. The (difluoroamino)silane has promise as a new synthon for the introduction of the -NF2 group into a variety of electrophilic inorganic and organic substrates. Activation barriers and relative energies were calculated for the unimolecular decompositions of Me3SiCF3 and t-Bu3SiNF2 using density functional theory (B3LYP/6-31G). The calculated activation energies confirm the long-assumed kinetic stability of Me3SiCF3.     

Intense, broadband, pulsed I-R source at the national synchrotron light source

We describe a broadband (1 m–1 mm) synchrotron radiation infrared source, pulsed each 20–180 nseconds and delivering about 10¹⁵ photons/sec/1% bandpass into f10 optics. The source size is diffraction limited. This source is thus 100–1000 times brighter than a 2000°K black body, very stable and capable of being used for calibration.Work supported by the U.S. Department of Energy.     

The use of semiempirical energy partitioning terms in the study of through space (homoaromatic) interactions

MNDO and AMI calculations, including configuration interactions, were performed on cycloheptatriene (2), 1,6-methano[10]annulene (3), and the tautomeric elassovalenes (4), (5), and (6). The goal of this study is to examine these systems and assess indicators of the importance of through space (homoaromatic) interactions. It is established that the two-center energy partitioning terms are capable of detecting favorable (negative two-center term) through space interactions. In cases of cyclic conjugation (homoconjugation), it is also shown that the inclusion of CI is necessary.     

A high pressure thermobalance

A high pressure thermobalance was assembled by placing a DuPont Model 950 balance into a stainless steel enclosure. The thermobalance is capable of operation to a maximum pressure (of N₂) to 500 atm and to a maximum temperature of 500°C. Operation of the instrument is illustrated by the thermogravimetric curves of BaBr₂·2H₂O, CuSO₄·5H₂0 and NaHCO₃ at various pres