Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells

Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP(3) binding pocket for enhanced IP(3) selectivity and for rapid and direct visualization of intracellular IP(3) > or = 0.5 microM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP(3) production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP(3) probe was extremely effective to quantitatively assess real-time physiological IP(3) production via those pathways formed only in the intact cellular configuration.     

Sample enrichment by using monolithic precolumns in microcolumn liquid chromatography

An on-line sample enrichment system was designed using monolithic precolumns in microcolumn LC. The monolithic ODS capillary columns were prepared via in situ sol-gel processes. The enrichment efficiency of the monolithic columns was tested by using phthalates as the analytes. The relative standard deviations (n = 6) for the retention time, peak area and peak height were between 0.4 and 1.2%, 0.9 and 5.5% and 0.4 and 3.9%, respectively. The system was linear (R2 > 0.99) within the working sample concentration and sample volume ranges. Comparing to 0.2 microl injection with a typical sample injector, the theoretical plate number of a same separation column was increased by 3-6-fold when the precolumn unit was used for sample injection. The recoveries of the analytes were between 88 and 120%, and the sample volume that could be injected into the system was increased up to 5000-fold. The limits of detection were improved by more than 2000-fold and were between 0.21 and 0.87 ng ml(-1) even with a UV absorbance detector. This system was applied to the determination of phthalates contained in laboratory distilled water and tap water samples.     

Fluorometric detection with a packed flow cell in micro high-performance liquid chromatography

Summary A packed flow cell was used for fluorometric detection in micro high-performance liquid chromatography. The flow cell consisted of fused-silica tubing packed with the same material as the separation column. A focusing effect of the stationary phase on the signal intensity was observed, leading to an improvement of the mass detection limit, as achieved by on-column detection.     

Spongistatin synthetic studies. An efficient, second-generation construction of an advanced ABCD intermediate

[reaction: see text] A short, efficient, and stereocontrolled synthesis of (-)-4, an advanced ABCD subunit of the spongistatins, has been achieved. Central to the synthetic strategy is the multicomponent linchpin union of silyl dithianes with epoxides to access both the AB and CD fragments. Fragment coupling was then achieved via an efficient stereoselective aldol reaction. The linear sequence required 22 steps and proceeded in 4.0% overall yield.     

Infrared studies of the less stable cis form of N-methylformmaide and N-methylacetamide in low-temperature nitrogen matrices and vibrational analyses of the trans and cis forms of these molecules

Recently studies of less stable rotational isomers became possible using a combination of low-temperature matrix-isolation infrared spectroscopy and either the high-temperature nozzle technique or irradiation with ultraviolet or near-infrared light. In this paper the infrared spectra of the less stable cis form of N-methylformamide and N-methylacetamide and their variously deuterated species were obtained in low-temperature nitrogen matrices using the high-temperature nozzle technique. The assignments of the bands due to the cis form as well as the trans form were made on the basis of normal-coordinate calculations.     

Bromination by means of sodium monobromoisocyanurate (SMBI)

A variety of aromatic compounds with both activating and deactivating substituents were brominated with sodium monobromoisocyanurate (SMBI) 1, diethyl ether, diethyl ether-methanesulfonic acid, trifluoroacetic acid, or sulfuric acid were employed as solvents. Thus nitrobenzene was conveniently brominated in sulfuric acid, benzene was readily monobrominated in diethyl ether-methanesulfonic acid, and phenol was selectively brominated at the ortho position under mild conditions in refluxing diethyl ether. With substituents that are easily protonated, trifluoroacetic acid may be employed as solvent in the reaction with 1, in contrast NBS was ineffective in trifluoroacetic acid. This renders 1 a superior reagent relative to NBS. In addition to aromatics, alkenes, ketones and esters were also brominated with 1. Diethyl malonate was brominated with 1 and then subjected to a Bingel reaction with NaH to afford the desired methanofullerene in reasonable yield.     

Reactivity of the indole ring in palladium(II) complexes of 2N1O-donor ligands: cyclopalladation and pi-cation radical formation

The Pd(II) complexes of new 2N1O-donor ligands containing a pendent indole, 3-[N-2-pyridylmethyl-N-2-hydroxy-3,5-di(tert-butyl)benzylamino]ethylindole (Htbu-iepp), 1-methyl-3-[N-2-pyridylmethyl-N-2-hydroxy-3,5-di(tert-butyl)benzylamino]ethylindole (Htbu-miepp), 3-[N-2-pyridylmethyl-N-2-hydroxy-3,5-di(tert-butyl)benzylamino]methylindole (Htbu-impp), and 3-(N-2-pyridylmethyl-N-4-hydroxybenzylamino)ethylindole (Hp-iepp) (H denotes a dissociable proton), were synthesized, and the structures of [Pd(tbu-iepp)Cl] (1a), [Pd(tbu-iepp-c)Cl] (1b), [Pd(tbu-miepp)Cl] (3), and [Pd(p-iepp-c)Cl] (4) (tbu-iepp-c and p-iepp-c denote tbu-iepp and p-iepp bound to Pd(II) through a carbon atom, respectively) were determined by X-ray analysis. Complexes 1a prepared in CH(2)Cl(2)/CH(3)CN and 3 prepared in CH(3)CN have a pyridine nitrogen, an amine nitrogen, a phenolate oxygen, and a chloride ion in the coordination plane. Complex 1b prepared in CH(3)CN has the same composition as 1a and was revealed to have the C2 atom of the indole ring bound to Pd(II) with the Pd(II)-C2 distance of 1.973(2) A. The same Pd(II)-indole C2 bonding was revealed for 4. Interconversion between 1a and 1b was observed for their solutions, the equilibrium being dependent on the solvent used. Reaction of 1b and 4 with 1 equiv of Ce(IV) in DMF gave the corresponding one-electron-oxidized species, which exhibited an ESR signal at g = 2.004 and an absorption peak at approximately 550 nm, indicating the formation of the Pd(II)-indole pi-cation radical species. The half-life, t(1/2), of the indole radical species at room temperature was calculated to be 20 s (k(obs) = 3.5 x 10(-)(2) s(-)(1)) for 1b. The cyclic voltammogram for 1b in DMF gave two irreversible oxidation peaks at E(pa) = 0.68 and 0.80 V (vs Ag/AgCl), which were ascribed to the oxidation processes of the coordinated indole and phenolate moieties, respectively.     

Synthesis of Ultrafine SiC Powders from Carbon-Silica Hybridized Precursors with Carbothermic Reduction

Inorganic-organic hybrid gels were prepared by simultaneous condensation of the liquid mixture of ethyl silicate, ethyl borate and water soluble phenol resin with low molecular weight. The liquid mixtures were changed into lightly colored transparent gels after a catalyst addition and stirring. The obtained gels were crushed, dried and fired in controlled conditions to yield the inorganic precursors. Since the tailored inorganic precursors mainly consisted of SiO2 and carbon, silicon carbide was formed with the heat treatments in an Ar atmosphere beyond 1773 K with carbothermic reduction. The properties and morphology of the formed silicon carbide powders in terms of the starting precursor compositions and the conditions of the carbothermic reduction were investigated with SEM, XRD and TG-DTA analysis.     

trans-2,2'-Bi(1-phenyladamantylidene): the most twisted biadamantylidene

The Grignard coupling of 2,2-dibromo-1-phenyladamantane gave trans-2,2'-bi(1-phenyladamantylidene) (1-Ph). Single-crystal X-ray analysis indicated that 1-Ph has a 23.2 degrees twisted double bond, which is much more distorted than that of parent 2,2'-biadamantylidene (1-H) and that of the ethyl-substituted derivative (1-Et). A cyclic voltammogram showed a reversible electron oxidation wave at 0.87 V vs Fc/Fc(+), which is 0.19 V lower than 1-H, indicating a significant increase in the HOMO energy level due to the distortion. The reaction of 1-Ph with 0.9 equiv of bromine gave an intramolecular Friedel-Crafts alkylation product, while bromination of 1-H and 1-Me has been reported to give a bridged bromonium ion and a rearranged product, 2-(1-methyl-2-adamantylidene)-4-bromotricyclo[5,3,1,0(3.9)]undec-4-ene, respectively.