Simultaneous determination of inositol and inositol phosphates in complex biological matrices: quantitative ion‐exchange chromatography/tandem mass spectrometry

myo‐Inositol (Ins) and myo‐inositol phosphates (InsPs) are widely distributed in plants and animals. The evaluation of the distribution of Ins and InsPs in cells and plant sources can impact the understanding of their role in nutrition, cellular processes and diseases, and how they may be modulated by diet. We developed an anion‐exchange chromatography/tandem mass spectrometry (HPLC/ESI‐MS/MS) method for the separation and simultaneous quantitation of Ins and different naturally occurring phosphorylated inositol compounds. Chromatographic separation was achieved in 30 min on a commercial anion‐exchange column (0.5 × 150 mm) using a gradient of 200 mM ammonium carbonate buffer (pH 9.0) and 5% methanol in H₂O. Analytes were identified by selective reaction monitoring using a triple quadrupole mass spectrometer in negative ion electrospray ionization mode. Adenosine 5′‐monophosphate was used as a general internal standard for quantitation. Detection is linear in the range of 0.25–400 pmol for Ins, InsP₁, InsP₄, and InsP₅, 40–400 pmol for InsP₂ and InsP₃, and 60–400 pmol for InsP₆, with a minimum r² > 0.994. The limit of detection is 0.25 pmol with a signal‐to‐noise ratio of 10:1 for all analytes. The intra‐day and inter‐day variations were within 17% at three concentration levels. Recovery values for the seven analytes spiked into extraction solution or different matrices were between 63 and 121%. Using this approach, Ins and InsPs were measured in three different plant samples and in cultured cells, illustrating significant differences in the distribution of inositol compounds in food samples compared to cells and between cell types. Copyright © 2009 John Wiley & Sons, Ltd.     

Spin-cluster effects and frustration in the uniaxial spin glass Fe2−xTi1+xO5

Transverse-field (TF) muon-spin-relaxation (μSR) and Mössbauer experiments on the uniaxial insulating spin glass Fe_2?xTi_1+xO₅ (x=0.25) have been performed near and below the spin-glass temperature (T _g). The effect of a transverse field on the spin-freezing process and spin-glass state has been investigated by measuring the field-and temperature-dependencies of the μSR parameters. Spin-cluster effects signaled by anomalous μ-spin relaxation have been observed in a temperature region just aboveT _g. An interpretation supported by recently developed theoretical models addressing non-linear relaxation in an intermediate Griffiths phase is offered.     

Depurinating acylfulvene-DNA adducts: characterizing cellular chemical reactions of a selective antitumor agent

Acylfulvenes (AFs) are a class of semisynthetic agents with high toxicity toward certain tumor cells, and for one analogue, hydroxymethylacylfulvene (HMAF), clinical trials are in progress. DNA alkylation by AFs, mediated by bioreductive activation, is believed to contribute to cytotoxicity, but the structures and chemical properties of corresponding DNA adducts are unknown. This study provides the first structural characterization of AF-specific DNA adducts. In the presence of a reductive enzyme, alkenal/one oxidoreductase (AOR), AF selectively alkylates dAdo and dGuo in reactions with a monomeric nucleoside, as well as in reactions with naked or cellular DNA, with 3-alkyl-dAdo as the apparently most abundant AF-DNA adduct. Characterization of this adduct was facilitated by independent chemical synthesis of the corresponding 3-alkyl-Ade adduct. In addition, in naked or cellular DNA, evidence was obtained for the formation of an additional type of adduct resulting from direct conjugate addition of Ade to AF followed by hydrolytic cyclopropane ring-opening, indicating the potential for a competing reaction pathway involving direct DNA alkylation. The major AF-dAdo and AF-dGuo adducts are unstable under physiologically relevant conditions and depurinate to release an alkylated nucleobase in a process that has a half-life of 8.5 h for 3-alkyladenine and less than approximately 2 h for dGuo adducts. DNA alkylation further leads to single-stranded DNA cleavage, occurring exclusively at dGuo and dAdo sites, in a nonsequence-specific manner. In AF-treated cells that were transfected with either AOR or control vectors, the DNA adducts identified match those from in vitro studies. Moreover, a positive correlation was observed between DNA adduct levels and cell sensitivity to AF. The potential contributing roles of AOR-mediated bioactivation and adduct stability to the cytotoxicity of AF are discussed.     

Thiazole orange-peptide conjugates: sensitivity of DNA binding to chemical structure

[structure: see text] Derivatives of the highly fluorescent and DNA-binding dye thiazole orange (TO) are described that feature appended peptides. Functionalization of TO can be achieved at either of the endocyclic nitrogens, and the photophysical properties and DNA-binding modes are sensitive to the position of the tethered peptide. A series of TO-peptide conjugates are described, demonstrating the utility of a solid-phase synthesis approach to their preparation and illustrating how the photophysical and DNA-binding properties of the compounds are influenced by chemical structure.     

One-step synthesis and characterization of difunctionalized N-confused tetraphenylporphyrins

Three disubstituted N-confused porphyrins (2-4) were prepared in ca. 4% yield using a one-pot synthesis. These porphyrins bear 3,5-di-tert-butylphenyl groups substituted at the C(5) and C(20) meso positions and para-substituted (Br, NO(2), ethynyl) phenyl groups at the C(10) and C(15) meso positions. The specific orientation of the aryl rings around the macrocycle in porphyrin 2 was definitively determined using a combination of 1D ((1)H and (13)C) and 2D (gHMQC and gHMBC) NMR spectroscopy. The absorption spectra of 2-4 in CH(2)Cl(2) and dimethylacetamide are similar to those of N-confused tetraphenylporphyrin in the same solvents but have Soret and Q-bands that are shifted to lower energies. Steady-state fluorescence measurements revealed Q(x)(0,0) and Q(x)(0,1) bands similar in energy to the unsubstituted NCPs 1i and 1e. The fluorescence quantum yield results for two of these NCPs (2, 4) are atypical of porphyrin behavior and are being further investigated by time-resolved spectroscopy.     

Comparison of the quality of aqueous dispersions of single wall carbon nanotubes using surfactants and biomolecules

The use of single wall carbon nanotubes (SWCNTs) in current and future applications depends on the ability to process SWCNTs in a solvent to yield high-quality dispersions characterized by individual SWCNTs and possessing a minimum of SWCNT bundles. Many approaches for the dispersion of SWCNTs have been reported. However, there is no general assessment which compares the relative quality and dispersion efficiency of the respective methods. Herein we report a quantitative comparison of the relative ability of "wrapping polymers" including oligonucleotides, peptides, lignin, chitosan, and cellulose and surfactants such as cholates, ionic liquids, and organosulfates to disperse SWCNTs in water. Optical absorption and fluorescence spectroscopy provide quantitative characterization (amount of SWCNTs that can be suspended by a given surfactant and its ability to debundle SWCNTs) of these suspensions. Sodium deoxy cholate (SDOCO), oligonucleotides (GT)(15), (GT)(10), (AC)(15), (AC)(10), C(10-30), and carboxymethylcellulose (CBMC-250K) exhibited the highest quality suspensions of the various systems studied in this work. The information presented here provides a good framework for further study of SWCNT purification and applications.     

2 + 2 Dimerization of cinnamylidene malonic acid: A structural and kinetic study

Cinnamylidene malonic acid was synthesized and its crystal structure obtained. The dicarboxylic acid hydrogen-bonding motif of this structure consists of catemer chains that lie along the c- glide plane. A photo induced 2?+?2 cycloaddition occurred upon exposure of the crystals to UV light which resulted in the destruction of the crystal. The structure of the photo products were determined by IR and NMR analysis. The regio- and stereo-chemistry of the photoproduct can be rationalized by examining the relative orientation and symmetry relating reacting molecules within the crystal structure. A kinetic study demonstrated first order reaction kinetics which is consistent with a reaction occurring under topochemical control.     

Combinatorial Probes for High‐Throughput Electrochemical Analysis of Circulating Nucleic Acids in Clinical Samples

The analysis of circulating tumour nucleic acids (ctNAs) provides a minimally invasive way to assess the mutational spectrum of a tumour. However, effective and practical methods for analyzing this emerging class of markers are lacking. Analysis of ctNAs using a sensor‐based approach has notable challenges, as it is vital to differentiate nucleic acids from normal cells from mutation‐bearing sequences emerging from tumours. Moreover, many genes related to cancer have dozens of different mutations. Herein, we report an electrochemical approach that directly detects genes with mutations in patient serum by using combinatorial probes (CPs). The CPs enable detection of all of the mutant alleles derived from the same part of the gene. As a proof of concept, we analyze mutations of the EGFR gene, which has more than 40 clinically relevant alterations that include deletions, insertions, and point mutations. Our CP‐based approach accurately detects mutant sequences directly in patient serum.     

A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria

The measurement of physicochemical parameters in living cells can provide information on individual cellular organelles, helping us to understand subcellular function in health and disease. While organelle‐specific chemical probes have allowed qualitative evaluation of microenvironmental variations, the simultaneous quantification of mitochondrial local microviscosity (η_m) and micropolarity (?_m), along with concurrent structural variations, has remained an unmet need. Herein, we describe a new multifunctional mitochondrial probe ( MMP ) for simultaneous monitoring of η_m and ?_m by fluorescence lifetime and emission intensity recordings, respectively. The MMP enables highly precise measurements of η_m and ?_m in the presence of a variety of agents perturbing cellular function, and the observed changes can also be correlated with alterations in mitochondrial network morphology and motility. This strategy represents a promising tool for the analysis of subtle changes in organellar structure.