Serum Chloride Optical Sensors Based on Dynamic Quenching of the Fluorescence of Photo-Immobilized Lucigenin

This article describes the preparation and performance of an optical sensor for continuous measurement of chloride at extracellular (serum) levels (20–200mM). The sensor is based on dynamic quenching of the fluorescence of lucigenin which was photo-immobilized on a hydrogel. Quenching occurs via a collisional mechanism, and the decrease in fluorescence intensity on exposure to 100mM chloride typically is –60%. It allows the determination of chloride in the 1 to 200mM concentration range, with a precision of ±3mM at 120mM. Bromide, iodide and salicylate act as interferents, while the effect of pH and oxygen is negligibly small. The sensor displays strong fluorescence intensity, excellent reproducibility, long-term stability, response times in the order of 2–5min, and is used in a commercial serum and blood chloride analyzer.Received January 21, 2003; accepted April 6, 2003 Published online July 28, 2003     

A 1,8-naphthalenediol-based unsymmetrical dinucleating ligand

A facile synthesis of 2-formyl-1,8-naphthalenediol is reported. Its potential as a general precursor for the preparation of unsymmetrical multidentate chelating ligand systems based on 1,8-naphthalenediol is demonstrated by the synthesis of the dinucleating ligand L(4-)(H(4)L=N,N'-bis(2-(1,8-naphthalenediol)methylidene)propylenediamine). Reaction of H(4) L with copper acetate results in the formation of the unsymmetrical dinuclear Cu(II) complex [LCu(2)](3), which has been structurally characterized by single-crystal X-ray diffraction. One Cu(II) ion is coordinated by a N(2)O(2) compartment of L(4-) and the other Cu(II) ion is coordinated by an O(4) compartment of L(4-) while they are bridged by two aryloxide functions of L(4-). A dimerization of two molecules of 3 to a tetranuclear entity 3(2) occurs through formation of weak apical Cu--O interactions. Analysis of the temperature dependent magnetic susceptibility measurements (2--290 K) established a strong intradimer exchange coupling J(12)=-371 cm(-1). This strong superexchange interaction fits nicely in a magneto-structural correlation which has been established for dinuclear bis(phenoxide)-bridged Cu(II) complexes demonstrating the electronic equivalence of the aryloxides of a phenol and 1,8-naphthalenediol.     

Biosynthesis of isoprenoids. purification and properties of IspG protein from Escherichia coli

[Structure: see text]. The IspG protein is known to catalyze the transformation of 2-C-methyl-d-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in the nonmevalonate pathway of isoprenoid biosynthesis. We have found that the apparent IspG activity in the cell extracts of recombinant Escherichia coli cells as observed by a radiochemical assay can be enhanced severalfold by coexpression of the isc operon which is involved in the assembly of iron-sulfur clusters. The recombinant protein was isolated by affinity chromatography under anaerobic conditions. With a mixture of flavodoxin, flavodoxin reductase, and NADPH as the reducing agent, stringent assay methods based on photometry or on 13C NMR detection of multiply 13C-labeled substrate/product ratios afforded catalytic activities greater than 60 nmol mg(-1) min(-1) for the protein "as isolated" (i.e., without reconstitution of any kind). Lower apparent activities were found using photoreduced deazaflavin as an artifactual electron donor, whereas dithionite was unable to serve as an artificial electron donor. The apparent Michaelis constant for 2-C-methyl-D-erythritol 2,4-cyclodiphosphate was 700 microM. The enzyme was inactivated by EDTA and could be reactivated by Mn2+. The pH optimum was at 9.0. The protein contained 2.4 iron ions and 4.4 sulfide ions per subunit. The replacement of any of the three conserved cysteine residues afforded mutant proteins which were devoid of catalytic activity and contained less than 6% of Fe2+ and less than 23% of S2- as compared to the wild-type protein. Sequence comparison indicates that putative IspG proteins of plants, the apicomplexan protozoan Plasmodium falciparum, and bacteria from the Bacteroidetes/Chlorobi group contain an insert of about 170-320 amino acid residues as compared with eubacterial enzymes.     

Synthesis of functionalized 2-alkylidenetetrahydrofurans by cyclization of 1,3-bis(trimethylsilyloxy)-1,3-butadienes with epoxides

The Lewis acid mediated cyclization of epoxides with 1,3-bis(trimethylsilyloxy)-1,3-butadienes, electroneutral equivalents of 1,3-dicarbonyl dianions, results in the formation of 2-alkylidenetetrahydrofurans with a great variety of substitution patterns and functional groups. This includes the synthesis of 2,3'-bifuranylidenes and 7-oxabicyclo[4.3.0]nonanes. The cyclization of dienes with functionalized epoxides containing base-labile groups proceeds with good chemoselectivity. In all reactions, good regio- and E diastereoselectivities are observed. Based on the stereoselectivities observed for reactions of 1,2-disubstituted epoxides, a working hypothesis for the mechanism of the reaction is suggested.     

Structural requirements in chiral diphosphine-rhodium complexes. VIII. Asymmetric hydrogenation of N-acetyldehydroamino acids with rhodium(I) complexes containing chiral carbocyclic analogues of DIOP.

Z--acetamidocinnamic acid was hydrogenated with neutral diphosphine-rhodium(I) complexes containing trans-1,2-bis(diphenylphosphinomethyl) cycloalkanes to give N-acetylphenylalanine: 86 % e.e.-(R) [(1R,2R)-cyclobutane]; 63 % e.e.-(R) [(1R,2R)-cyclopentane]; 35 % e.e.-(S) [(1S,2S)-cyclohexane]; and 82 % e.e.-(R) [(2R,3R)-DIOP]. Similarly, -acetamidoacrylic acid was hydrogenated to give N-acetylalanine: 72 % e.e.-(R) [(1R,2R)-cyclobutane]; 72 % e.e.-(R) [(1R,2R)-cyclopentane]; 40 % e.e.-(S) [(1S,2S)-cyclohexane]; and 73 % e.e.-(R) [(2R,3R)-DIOP].     

Implications of active site constraints on varied DNA polymerase selectivity

DNA polymerase selectivity often varies significantly depending on the DNA polymerase. The origin of this varying error propensity is elusive. It is assumed that DNA polymerases form nucleotide binding pockets that differ in properties such as shape and tightness. We tested this prediction and studied HIV-1 RT by employment of size-augmented nucleotides and site-directed mutagenesis of the enzyme. New valuable insights into the mechanism of DNA polymerase fidelity were obtained. The presented study provides experimental evidence that variations of steric constraints within the nucleotide binding pocket of at least two DNA polymerases cause variations in nucleotide incorporation selectivity. Thus, our results support the concept of active site tightness as a causative in differential fidelity among DNA polymerases.     

Electrospray ionization mass spectrometric studies of nucleophilic carbenes derived from pyrazolium and indazolium carboxylates

Pyrazolium-3-carboxylate and indazolium-3-carboxylate, which belong to the class of pseudo-cross-conjugated mesomeric betaines and which represent the electronically relevant partial structures of the betaine alkaloid Nigellicin, were examined by electrospray ionization mass spectrometry. These compounds decarboxylate to pyrazol-3-ylidene and indazol-3-ylidene. The formation of adducts of these new nucleophilic carbenes under the measurement conditions was examined.     

[(Mes3Sn)2MoO4], ein monomeres Triorganozinnmolybdat

[(Mes₃Sn)₂MoO₄], a Monomeric Triorganotin Molybdate Mes₃SnBr (Mes = 1, 3, 5‐trimethylphenyl) reacts with (NBu₄)₂[Mo₆O₁₉] in the presence of (NBu₄)OH (in CH₃CN as solvent) to form [(Mes₃Sn)₂MoO₄]. Alternatively the title compound can be obtained from the reaction of [MoO₂(acac)₂] (acac = 2, 4‐pentadionate) with Mes₃SnOH in isopropanol. [(Mes₃Sn)₂MoO₄] forms monoclinic crystals, space group C2/c, with a = 2271.6(3) pm, b = 825.2(1) pm, c = 2739.9(5) pm, β = 90.96(2)°. The crystal structure consists of isolated molecules in which a tetrahedral MoO₄ unit is connected to two terminal Mes₃Sn groups. The Mo‐O distances range from 169.6(4) to 181.1(3) pm and the Sn‐O distance is 204.8(3) pm.