bluuebunny/crossref_metadata_2025 huggingface dataset:
Solution‐Phase Synthesis of Di‐ and Trinucleotides Using Polymer‐Supported Reagents
Authors: Morvan François , Vasseur Jean‐Jacques
Published: September 2006

A solution‐phase phosphoramidite synthesis of short oligonucleotides using solid‐supported reagents is described. Polyvinyl pyridinium tosylate is employed as the activator in the coupling step between nucleoside‐3′‐O‐phosphoramidites and 5′‐OH nucleosides. The resulting dinucleotide phosphite triesters are oxidized or sulfurized with polymer‐bound periodate or tetrathionate to phosphotriesters or thiono phosphotriesters, respectively. Removal of the 3′‐O‐levulinyl protection is accomplished with a polymer‐supported hydrazine. In this approach, the excess reagents are removed by filtration, avoiding complicated purifications. All the polymer‐bound reagents used in this method result from salt bridge interactions with the polymers. Consequently, the resins are regenerable.
DNA Synthesis Without Base Protection Using the Phosphoramidite Approach
Authors: Ohkubo Akihiro , Seio Kohji , Sekine Mitsuo
Published: September 2006

In the strategy of DNA synthesis without base protection, a recentO‐selective phosphorylation procedure known as the “activated phosphite method” is used to generate reactive phosphite intermediates fromN‐unprotected phosphoramidite building blocks. This new strategy proved to be advantageous over previous strategies because the cleavage of the N‐P(III) bond, which can be formed by the significant phosphitylation of the nucleobase amino groups, can be omitted. The activated phosphite method provides an attractive route to high‐throughput synthesis of DNA and the synthesis of alkali‐labile modified DNAs such as oligonucleotides containing DNA lesions. This unit presents two convenient methods for the synthesis ofN‐unprotected deoxynucleoside 3′‐phosphoramidite units and detailed procedures for automated synthesis of oligodeoxyribonucleotides from these phosphoramidite units.
Mass Determination of Phosphoramidites
Authors: Harusawa Shinya , Fujitake Mihoyo , Kurihara Takushi , Zhao Zheng‐yun , Lilley David M.J.
Published: September 2006

Nucleoside phosphoramidites are the most widely used building blocks in contemporary solid‐phase synthesis of oligonucleotides. The accurate molecular weight measurements of such molecules, which are acid‐labile compounds, may be easily determined by mass spectrometry using a matrix system, triethanolamine/NaCl, on a liquid secondary ion mass spectrometer (LSIMS) or fast‐atom bombardment (FAB) MS equipped with a double‐focusing mass spectrometer. The present method rapidly and easily measures the accurate molecular weights of various phosphoramidites as adduct ions [M+Na]+ with an average mass error smaller than 0.4 ppm, allowing determination of the formulas of the phosphoramidites in place of elemental analysis. Further, it was found that intensities of molecular‐related ions could be enhanced to the highest degree by adjustment of the molar ratio of phosphoramidite and NaCl, fixing the amount of triethanolamine on LSIMS, making the present method a powerful tool for identification of phosphoramidites by mass spectrometry.
Chemical Phosphorylation of Deoxyribonucleosides and Thermolytic DNA Oligonucleotides
Authors: Ausín Cristina , Grajkowski Andrzej , Cieślak Jacek , Beaucage Serge L.
Published: September 2006

The phosphorylating reagent bis[S‐(4,4′‐dimethoxytrityl)‐2‐mercaptoethyl]‐N,N‐diisopropylphosphoramidite is prepared in three steps from commercial methyl thioglycolate and diisopropylphosphoramidous dichloride. The phosphorylating reagent has been used successfully in the solid‐phase synthesis of deoxyribonucleoside 5′‐/3′‐phosphate or ‐thiophosphate monoesters and oligonucleotide 5′‐phosphate/‐thiophosphate monoesters. Bis[S‐(4,4′‐dimethoxytrityl)‐2‐mercaptoethyl]‐N,N‐diisopropylphosphoramidite has also been employed in the construction of a thermolytic dinucleotide prodrug model to evaluate the ability of the reagent to produce thermosentive oligonucleotide prodrugs under mild temperature conditions (∼25°C) for potential therapeutic applications.
Production of Monoclonal Antibodies
Authors: Yokoyama Wayne M. , Christensen Michelle , Santos Gary Dos , Miller Diane
Published: September 2006

This unit describes the production of monoclonal antibodies beginning with immunization and cell fusion and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium. An alternate protocol describes cell fusion and one‐step selection and cloning of hybridomas utilizing a semi‐solid methylcellulose‐based medium (ClonaCell®‐HY).
Preparation and Coculture of Neurons and Glial Cells
Authors: Viviani Barbara
Published: September 2006

The close relationship between neurons, glia, astroglia, and astrocytes plays a relevant role in modulating a neurotoxic effect and propagating the consequent degenerative event in the brain. After exposure to a neurotoxicant, glial cells are often activated to release substances responsible for the propagation of the damage. the production of such mediators is the result of both neural death and communication between microglia and astrocytes. Because they are simple and highly controlled, cocultures of different cells of the nervous system are a valuable tool in the investigations of these complex relationships. This unit describes protocols to set up neuron‐glia and microglia‐astrocyte sandwich cocultures, in which the selected cell populations grow on two different surfaces. This system has the advantage that the two cell populations can be separated at any point in the exposure without disrupting the cells integrity and organization
Assays for Regulated Exocytosis of Mast Cell Granules
Authors: Blank Ulrich , Rivera Juan
Published: September 2006

Mast cells are important effectors in innate and adaptive immune responses. They contain numerous secretory granules filled with inflammatory mediators in their cytoplasm. Exocytosis of granular content does not take place until the cell receives an appropriate stimulus such as the aggregation of IgE antibody bound to high‐affinity IgE receptors by specific antigen. This process is therefore referred to as regulated exocytosis. A characteristic of mast cell exocytosis is that it does not involve release of a few individual granules, but rather, a large fraction of the granular content is released due to compound exocytosis, implicating the occurrence of granule‐to‐granule and granule‐to‐plasma membrane fusion. This unit describes assays that measure the release of granular content from mast cells. They include in vitro colorimetric‐, radiolabel‐, or antibody detection–based assays for substances stored in the granules. Given that animal models for basophil and mast cell activation are being used with increasing frequency, the unit also includes protocols to measure exocytosis of mast cell granule content in vivo.
An Introduction to Recognizing Functional Domains
Authors: Stormo Gary D.
Published: September 2006

This unit provides an overview of issues involved in domain recognition in protein and DNA sequences. It opens with a discussion of the two primary methods of domain representation, namely consensus sequences and alignment matrices (e.g., the log‐odds matrix). The unit continues with a brief overview of some of the resources available for identifying functional domains in nucleotide sequences (e.g., TRANSFAC). In addition, it reviews databases such as Pfam, InterPro and Blocks, which are available for protein analysis.
Using Weeder for the Discovery of Conserved Transcription Factor Binding Sites
Authors: Pavesi Giulio , Pesole Graziano
Published: September 2006

One of the greatest challenges facing modern molecular biology is the understanding of the complex mechanisms regulating gene expression. A fundamental step in this process requires the characterization of motifs involved in the regulation of gene expression at transcriptional and post‐transcriptional levels. In particular, transcription is modulated by the interaction of transcription factors with their corresponding binding sites. Weeder is a software package freely available for noncommercial users as a stand‐alone or Web‐based application for the automatic discovery of conserved motifs in a set of related DNA sequences from coregulated genes. The motifs found are likely to represent instances of binding sites for some common transcription factor regulating the genes of the set. The program has been designed to make its usage as simple as possible and to require very little prior knowledge about the length and conservation of the motifs to be found.
An Introduction to Modeling Structure from Sequence
Authors: Petsko Gregory A.
Published: September 2006

The underlying premise behind all attempts to determine a large number of diverse protein structures is that the total number of protein domain folds is much smaller, by many orders of magnitude, than the total number of sequences; in other words, many sequences adopt essentially the same fold. If the fold of a protein could be recognized from sequence information alone, then a complete database of all possible folds would allow the structure corresponding to any sequence to be modeled. The growth of structure determination has turned most biochemists and biologists into consumers of structural information. As the demand for such information continues to outstrip the supply, all aspects of structure modeling assume increasing importance. This unit provides an introduction to modeling structure from its sequence and surveys the currently available methods described in the subsequent units of this chapter.
Getting a Tree Fast: Neighbor Joining, FastME, and Distance‐Based Methods
Authors: Desper Richard , Gascuel Olivier
Published: September 2006

Neighbor Joining (NJ), FastME, and other distance‐based programs including BIONJ, WEIGHBOR, and (to some extent) FITCH, are fast methods to build phylogenetic trees. This makes them particularly effective for large‐scale studies or for bootstrap analysis, which require runs on multiple data sets. Like maximum likelihood methods, distance methods are based on a sequence evolution model that is used to estimate the matrix of pairwise evolutionary distances. Computer simulations indicate that the topological accuracy of FastME is best, followed by FITCH, WEIGHBOR, and BIONJ, while NJ is worse. Moreover, FastME is even faster than NJ with large data sets. Best‐distance methods are equivalent to parsimony in most cases, but become more accurate when the molecular clock is strongly violated or in the presence of long (e.g., outgroup) branches. This unit describes how to use distance‐based methods, focusing on NJ (the most popular) and FastME (the most efficient today). It also describes how to estimate evolutionary distances from DNA and proteins, how to perform bootstrap analysis, and how to use CLUSTAL to compute both a sequence alignment and a phylogenetic tree.
Using MSDchem to Search the PDB Ligand Dictionary
Authors: Dimitropoulos Dimitris , Ionides John , Henrick Kim
Published: September 2006

The PDB ligand dictionary is the chemical reference database of all the small building block molecules (e.g., amino acids, nucleic acids, and bound ligands) in the Protein Data Bank (PDB) referenced by a distinct three‐letter code identifier. Since PDB files have only three‐dimensional coordinate data, the role of the dictionary that of a reference resource for the actual chemical properties of small molecules, shared consistently across all PDB entries. The ligand dictionary is maintained in all sites of the Worldwide Protein Data Bank (wwPDB), the Research Collaboratory for Structural Bioinformatics (RCSB) in U.S., the Macromolecular Structure Database (MSD) in Europe, and the Protein Data Bank in Japan (PDBj), and it is exchanged on a regular basis. The MSD group at the European BioInformatics Institute (EBI) extends the dictionary into the MSDchem ligand database, which utilizes chemo‐informatics packages and incorporates additional curation work. MSDchem is publicly available on the Web through the MSDchem search system, the functionality of which is described in more detail in this unit.
Murine Models of Streptococcus pyogenes Infection
Authors: Roberts Samantha , Scott June R. , Husmann Linda K. , Zurawski Christine A.
Published: September 2006

This unit describes procedures for testing virulence of Streptococcus pyogenes in mice. S. pyogenes is an important human pathogen and causes one of the most common childhood diseases. The syndromes that result from S. pyogenes infection are diverse, ranging from mild, superficial throat or skin infection to severe, invasive disea/se that is often lethal. Thus, a greater understanding of the virulence factors of this bacterium and development of modalities to prevent or relieve the infections it causes are important. Since S. pyogenes is a strictly human pathogen (with the exception of a single strain), the value of all animal models is limited. This unit describes a model for long‐term throat colonization following the natural route of infection (inhalation), one for pneumonia and systemic dissemination following intratracheal inoculation, and one for systemic dissemination following the more natural route of skin infection. In addition, methods are presented for culturing S. pyogenes from tissues of the infected animal.
A new approach for in vitro regeneration of tomato plants devoid of exogenous plant growth hormones
Authors: Plana Dagmara , Fuentes Alejandro , Alvarez Marta , Lara Regla M. , Alvarez Félix , Pujol Merardo
Published: September 2006

Many available methodologies for in vitro regeneration of commercial tomato varieties promote not only the production of normal shoots but also individual leaves, shoots without apical meristems and vitrified structures. All these abnormal formations influence and diminish the regeneration efficiency. At the basis of this phenomenon lies callus development. We optimized an alternative procedure by which the regeneration occurs without abnormal shoot formation. The portion including the proximal part of hypocotyls and the radicle was cultured on medium consisting of Murashige and Skoog salts, 4 mg/L thiamine, 100 mg/L mio‐inositol and 3% sucrose. After two‐three weeks, 60% explants showed adventitious shoot formation. No changes in the morphological characteristics of regenerated plants and fruits were observed as compared with parents. Karyotypic analysis of regenerated plants showed no variations in chromosome number. The optimized procedure offers the advantage of tomato plant regeneration avoiding callus formation, which enables normal plant recovery with an efficiency ranging from 1.45 ± 0.05 to 2.57 ± 0.06 shoots per explant in Campbell‐28, Amalia, Lignon, and Floradel cultivars.
New insights into mechanisms of growth and β‐carotene production in Blakeslea trispora
Authors: Böhme Karen , Richter Christin , Pätz Reinhard
Published: September 2006

Blakeslea trispora (Mucorales) has economic importance because of its ability to produce large amounts of β‐carotene. To shed light on the actual point of induction and to shorten the following production process, germination and growth of its two mating types, (–) and (+), were observed separately, and the mating point was investigated in lab scale experiments. The (–) mating type showed much faster germination than the (+) type on solid medium and in Erlenmeyer flasks. However, after a first period, the (–) mating type grew clearly slower than the (+) type. In addition, the (–) type branched more vividly than the (+) type. A ratio of 30:1 of (–) and (+) type at an age of 20 h was found to achieve highest β‐carotene yields. Our results provide a comprehensive overview of Blakeslea trispora growth and its product synthesis.
Expression of green fluorescent protein in pollen of oilseed rape (Brassica napus L.) and its utility for assessing pollen movement in the field
Authors: Moon Hong S. , Halfhill Matthew D. , Hudson Laura C. , Millwood Reginald J. , Stewart C. Neal
Published: September 2006

Transgene movement via pollen is an important component of gene flow from transgenic plants. Here, we present proof‐of‐concept studies that demonstrate the monitoring of short distant movement of pollen expressing a genetically encoded fluorescent tag in oilseed rape (Brassica napus L. cv. Westar). Transgenic oilseed rape plants were produced using Agrobacterium‐mediated transformation method with the pBINDC1 construct containing a green fluorescent protein (GFP) variant, mGFP5‐ER, under the control of the pollen‐specific LAT59 promoter from tomato. Transgenic pollen was differentiated from non‐transgenic pollen in vivo by a unique spectral signature, and was shown to be an effective tool to monitor pollen movement in the greenhouse and field. GFP‐tagged pollen also served as a practical marker to determine the zygosity of plants. In a greenhouse pollen flow study, more pollen was captured at closer distances from the source plant plot with consistent wind generated by a fan. Under field conditions, GFP transgenic pollen grains were detected up to a distance of 15 m, the farthest distance from source plants assayed. GFP‐tagged pollen was easily distinguishable from non‐transgenic pollen using an epifluorescence microscope.
Evaluation of anti‐methicillin‐resistant Staphylococcus aureus (MRSA) activity and synergy of some bioactive plant extracts
Authors: Aqil Farrukh , Ahmad Iqbal , Owais Mohd
Published: September 2006

Anti‐methicillin‐resistant Staphylococcus aureus (MRSA) activity of ethanolic extracts of four medicinal plants namely Acorus calamus (rhizome) Hemidesmus indicus (stem), Holarrhena antidysenterica (bark), and Plumbago zeylanica (root), were detected with inhibition zone size ranged from 11 to 44 mm and minimum inhibitory concentration (MIC) varied from 0.32 to 3.25 mg/mL. Further, ethyl acetate, acetone and methanol fractions of above plants demonstrated antibacterial activity. The potency of these fractions based on zone of inhibition and MIC value was relatively higher in P. zeylanica (ethylacetate fraction), followed by acetone fractions of H. indicus, A. calamus, and H. antidysenterica. Time kill assay with most promising fractions of these plant extracts, demonstrated concentration‐dependent killing of MRSA within 9–12 h of incubation. Interestingly, synergistic interaction among alcoholic extracts and some fractions of above four plants was evident against MRSA. Further, synergistic interaction of these extracts was detected with one or more antibiotics tested (tetracycline, chloramphenicol, ciprofloxacin, cefuroxime and ceftidizime). The findings also validate the traditional uses of above plants against infectious diseases. Phytochemical studies demonstrated flavonoids and phenols as major active constituents. Further investigations are needed to characterize the active principle and its interaction mechanism with antibiotics.
XVSAP1 from Xerophyta viscosa improves osmotic‐, salinity‐ and high‐temperature‐stress tolerance in Arabidopsis
Authors: Garwe Dahlia , Thomson Jennifer A. , Mundree Sagadevan G.
Published: September 2006

XVSAP1, a gene isolated from a dehydrated Xerophyta viscosa cDNA library, was transformed into Arabidopsis thaliana by Ti plasmid‐mediated transformation under the control of a cauliflower mosaic virus 35S promoter, a nos terminator and bar gene selection. Expression of XVSAP1 in Arabidopsis led to constitutive accumulation of the corresponding protein in the leaves. Transgenic Arabidopsis grown in tissue culture maintained higher growth rates during osmotic, high‐salinity and high temperature stress, respectively. Non‐transgenic plants had shorter roots, leaf expansion was inhibited and leaves were more chlorotic than those of the transgenic plants. This study demonstrates that XVSAP1 has a significant impact on dehydration, salinity and high‐temperature stress tolerance in Arabidopsis.
Use of virus vectors for the expression in plants of active full‐length and single chain anti‐coronavirus antibodies
Authors: Alamillo Josefa M. , Monger Wendy , Sola Isabel , García Beatriz , Perrin Yolande , Bestagno Marco , Burrone Oscar R. , Sabella Patricia , Plana‐Durán Joan , Enjuanes Luis , Lomonossoff George P. , García Juan A.
Published: September 2006

To extend the potential of antibodies and their derivatives to provide passive protection against enteric infections when supplied orally in crude plant extracts, we have expressed both a small immune protein (SIP) and a full‐length antibody in plants using two different plant virus vectors based on potato virus X (PVX) and cowpea mosaic virus (CPMV). The agr;SIP molecule consisted of a single chain antibody (scFv) specific for the porcine coronavirus, transmissible gastroenteritis virus (TGEV) linked to the α‐CH3 domain from human IgA. To express the full‐length IgA, the individual light and heavy chains from the TGEV‐specific mAb 6A.C3 were inserted into separate PVX constructs and plants were co‐infected with both constructs. Western blot analysis revealed the efficient expression of both the SIP and IgA molecules. Analysis of crude plant extracts revealed that both the plant‐expressed αSIP and IgA molecules could bind to and neutralize TGEV in tissue culture, indicating that active molecules were produced. Oral administration of crude extracts from antibody‐expressing plant tissue to 2‐day‐old piglets showed that both the αSIP and full‐length IgA molecules can provide in vivo protection against TGEV.
Production of biopharmaceuticals and vaccines in plants via the chloroplast genome
Authors: Daniell Henry
Published: September 2006

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi‐gene expression in a single transformation event. Hyper‐expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4–31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non‐green plastids (carrots, tomato), as well as the availability of antibiotic‐free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper‐expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon‐gamma (6% tsp), anti‐microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi‐subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast‐derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1‐ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.
Protein‐free transfection of CHO host cells with an IgG‐fusion protein: Selection and characterization of stable high producers and comparison to conventionally transfected clones
Authors: Lattenmayer Christine , Loeschel Martina , Schriebl Kornelia , Steinfellner Willibald , Sterovsky Thomas , Trummer Evelyn , Vorauer‐Uhl Karola , Müller Dethardt , Katinger Hermann , Kunert Renate
Published: September 2006

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein‐free transfection protocol for CHO cells, based on the Nucleofector™ technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum‐dependent cultivation. Our intensive characterization program was based on determination of specific productivity (qp) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 µg of DNA resulted in best productivities of the primary cell pools. After subcloning, the qp could be raised up to 27 pg · cells−1 · day−1. While the serum‐dependent transfectants exhibited specific productivities up to 57 pg · cells−1 · day−1 in serum‐dependent cultivation, a significant decrease that resulted in the range of qp of the protein‐free transfectants was observed after switching to protein‐free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein‐free adapted serum‐dependent transfectants. Therefore, we assume that problems during protein‐free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable‐producing recombinant CHO clones by protein‐free transfection of a protein‐free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein‐free transfection, a viable and highly efficient strategy for recombinant cell line development. Biotechnol. Bioeng. 2007;96:1118–1126. © 2006 Wiley Periodicals, Inc.
Sialic acid and oxidizability of lipid and proteins and antioxidant status in patients with coronary artery disease
Authors: Serdar Zehra , Yeşilbursa Dilek , Dirican Melahat , Sarandöl Emre , Serdar Akın
Published: September 2006

The aim of this study was to investigate the possible relationship between serum total sialic acid (TSA) concentration, recently shown to be a cardiovascular risk factor, and lipid and protein oxidation and antioxidant status and the severity of coronary artery disease (CAD) according to the obstructive vessel number in patients. The study was carried out on a total of 200 patients (142 men and 58 women) who were hospitalized for elective coronary angiographic evaluation with complaint of typical angina pectoris. According to the results of angiography, 150 patients had angiographically proven CAD (CAD group) and 50 patients had a history suggestive of angina pectoris but normal coronary angiograms (control group). The CAD group was further divided into single‐, double‐ and triple‐vessel disease groups according to the number of vessels involved. Lipid parameters were determined by routine laboratory methods. Plasma malondialdehyde (MDA) and vitamin E concentrations were determined by high‐performance liquid chromatography. TSA and other oxidant and antioxidant parameters were studied spectrophotometrically. Our results demonstrated significant increases both in TSA levels and in indicators of oxidative stress in the patients with CAD compared with the controls. However, antioxidant parameters were decreased in the patients with CAD. We found strong positive correlations between TSA and plasma MDA, Δ‐MDA which represents the degree of oxidative modification of apolipoprotein B‐containing lipoproteins, serum protein carbonyls and apolipoprotein B and weak correlations between TSA and low density lipoprotein cholesterol, triacylglycerol, paraoxonase, glutathione peroxidase (GPx), vitamin C and vitamin E. In conclusion, TSA is related to markers of lipid and protein oxidation, paraoxonase and GPx activities, vitamin C and E levels and the severity of CAD. Copyright © 2006 John Wiley & Sons, Ltd.
Modulation of Unconventional Fluorescence of Novel Photochromic Perimidine Spirodimers
Authors: Davis Riju , Tamaoki Nobuyuki
Published: September 2006

Fluorescence modulation by a class of photochromic perimidine spirodimers, which exhibit a characteristic fluorescence associated with their photochromic reactions, has been described. Upon irradiation using 365 nm light, these non‐fluorescent spiro molecules undergo a thermally‐reversible ring opening at their spiro junction resulting in the generation of strong fluorescence. The fluorescing species is distinctly different from both the stable ring‐closed and the ring‐opened compounds, though it appears to have been formed from and remains in equilibrium with the photochemically generated ring‐opened form. While the fluorescing species possesses a narrow absorption band with its maximum centered at 500 nm, the ring‐opened form exhibits a broad absorption across the visible region with two maxima centered at 410 and 650 nm, respectively. After initiating the photochromic reactions in these molecules using 365 nm light, purely photochemically‐controlled fluorescence modulation can be carried out using two wavelengths in the visible region, that is, 500 and 700 nm, while the equilibrium concentration of the ring‐opened form and the fluorescing species is controlled. Fluorescence modulation is attained also by controlling the ratio of the ring‐closed and ring‐opened forms by photochemical ring‐opening and thermal ring‐closing reactions. The study on the effect of substitution of these molecules suggests that by extending the conjugation of the perimidine core in the ring‐opened form the molecule is rendered non‐fluorescent and hence it can be assumed that the perimidine core forms the fluorescing entity of the molecule.
Synthesis of the First Chiral Bidendate Bis(trifluoromethyl)phosphane Ligand through Stabilization of the Bis(trifluoromethyl)phosphanide Anion in the Presence of Acetone
Authors: Hoge Berthold , Thösen Christoph , Pantenburg Ingo
Published: September 2006

Lewis acid/Lewis base adduct formation of the P(CF3)2− ion and acetone leads to a reduced negative hyperconjugation and, therefore, limits the CF bond activation. The resulting increased thermal stability of the P(CF3)2− ion in the presence of acetone allows selective substitutions and enables the synthesis of the first example of a chiral, bidentate bis(trifluoromethyl)phosphane ligand: a DIOP derivative, [(2,2‐dimethyl‐1,3‐dioxolane‐4,5‐diyl)bis(methylene)]bis(diphenylphosphane), in which the phenyl groups at the phosphorus atoms are replaced by strong electron‐withdrawing trifluoromethyl groups. The resulting high electron‐acceptor strength of the synthesized bidentate (CF3)2P ligand is demonstrated by a structural and vibrational study of the corresponding tetracarbonyl–molybdenum complex. The stabilization of the P(CF3)2− ion in the presence of acetone is based on the formation of a dynamic Lewis acid/Lewis base couple, (CF3)2PC(CH3)2O−. Although there is no spectroscopic evidence for the formation of the formulated alcoholate ion, the intermediate formation of (CF3)2PC(CH3)2O− could be proved through the reaction with (CF3)2PP(CF3)2, which yields the novel phosphane–phosphinite ligand (CF3)2PC(CH3)2OP(CF3)2. This ligand readily forms square‐planar Pt(II) complexes upon treatment with solid PtCl2.
Preparation of High‐Surface‐Area Zinc Oxide with Ordered Porosity, Different Pore Sizes, and Nanocrystalline Walls
Authors: Polarz Sebastian , Orlov Andrey V. , Schüth Ferdi , Lu An‐Hui
Published: September 2006

Transition‐metal‐oxide materials possessing ordered mesoporosity have recently attracted significant research interest due to their numerous potential applications. Among them, ordered mesoporous zinc oxide (ZnO) is a very tempting material because of the importance of ZnO in heterogeneous catalysis. Here, first results of the preparation of ordered mesoporous ZnO materials by using different templates are reported. Porous materials with high surface area, different pore sizes, and nanocrystalline ZnO walls were obtained. Furthermore, we compare the two fundamental templating techniques, involving liquid crystals or ordered mesoporous carbon materials as templates. Regarding the formation of mesoporous ZnO, it was evident that the hard‐matter carbon template is superior.
Photochemistry of Peroxoborates: Borate Inhibition of the Photodecomposition of Hydrogen Peroxide
Authors: Rey Sébastien , Davies D. Martin
Published: September 2006

The UV absorbance and photochemical decomposition kinetics of hydrogen peroxide in borate/boric acid buffers were investigated as a function of pH, total peroxide concentration, and total boron concentration. At higher pH borate/boric acid inhibits the photodecomposition of hydrogen peroxide (molar absorptivity and quantum yield of H2O2 and HO2−, (19.0±0.3) m−1 cm−1 and 1, and (237±7) m−1 cm−1 and 0.8±0.1, respectively). The results are consistent with the equilibrium formation of the anions monoperoxoborate, KBOOH=[H+][HOOB(OH)3−]/([B(OH)3][H2O2]), 2.0×10−8, R. Pizer, C. Tihal, Inorg. Chem. 1987, 26, 3639–3642, and monoperoxodiborate, KBOOB=[BOOB2−]/([B(OH)4−][HOOB(OH)3−]), 1.0±0.3 or 4.3±0.9, depending upon the conditions, with molar absorptivity, (19±1) m−1 cm−1 and (86±15) m−1 cm−1, respectively, and respective quantum yields, 1.1±0.1 and 0.04±0.04. The low quantum yield of monoperoxodiborate is discussed in terms of the slower diffusion apart of incipient .OB(OH)3− radicals than may be possible for .OH radicals, or a possible oxygen‐bridged cyclic structure of the monoperoxodiborate.
Efficient Synthesis of Chiral 1,1′‐Binaphthalenes by the Asymmetric Suzuki–Miyaura Reaction: Dramatic Synthetic Improvement by Simple Purification of Naphthylboronic Acids
Authors: Genov Miroslav , Almorín Antonio , Espinet Pablo
Published: September 2006

Naphthylboronic acids prepared as reported in the literature are contaminated with HCl. A very simple purification prior to their use in Suzuki–Miyaura couplings has been found to be crucial, rendering efficient some reactions that had been reported in the literature either to fail or to give extremely poor yields. With this improvement, parent boronic acids can be used instead of esters at moderate temperatures, and bromo derivatives can be used instead of iodo derivatives. Convenient access to chiral sterically hindered binaphthalene derivatives has been achieved through the use of boronic acids, bromonaphthalenes, and ferrocenylphosphane ligands. The products were obtained in good yields (95–55 %) and with good enantioselectivities (90–50 %). Bulkier ligands are less efficient in the coupling of hindered partners.
Dynamic Kinetic Resolution of Secondary Alcohols Combining Enzyme‐Catalyzed Transesterification and Zeolite‐Catalyzed Racemization
Authors: Zhu Yongzhong , Fow Kam‐Loon , Chuah Gaik‐Khuan , Jaenicke Stephan
Published: September 2006

Hydrophobic zeolite beta containing low concentrations of Zr or Al was found to be a good catalyst for the racemization of 1‐phenylethanol. The formation of styrene as a side product could be minimized by reducing the metal concentration in the zeolite beta. Combined with an immobilized lipase from Candida antarctica, the dynamic kinetic resolution of 1‐phenylethanol to the (R)‐phenylethylester can be achieved with high yield and selectivity. The reaction was best conducted in toluene as solvent at 60 °C, with higher temperatures leading to a loss in the enantioselectivity of the formed ester. By using high‐molecular‐weight acyl‐transfer reagents, such as vinyl butyrate or vinyl octanoate, a high enantiomeric excess of the product esters of 92 and 98 %, respectively, could be achieved. This is attributed to a steric effect: the bulky ester is less able to enter the pore space of the zeolite catalyst where the active sites for racemization are localized. Close to 100 % conversion of the alcohol was achieved within 2 h. If the more common acyl donor, isopropenyl acetate, was used, the enantiomeric excess (ee) of the formed ester was only 67 %, and the reaction was considerably slower.
Solid‐Phase Synthesis, Characterization, and Antibacterial Activities of Metallocene–Peptide Bioconjugates
Authors: Chantson Janine T. , Vittoria Verga Falzacappa Maria , Crovella Sergio , Metzler‐Nolte Nils
Published: September 2006

This work shows how the introduction of an organometallic group enhances and modifies the specificity of biologically active peptides. Ferrocene was chosen as an organometallic group because it has been shown to alter the pharmacodynamic profile of bioactive compounds. A comparison with the isosteric cobaltocenium group allows one to explore the influence of charge and redox potential on the biological activity of the conjugates. Arginine and tryptophan containing peptides H‐WRWRWR‐NH2 and H‐RWRWRW‐NH2 and the metallocene peptide bioconjugates [M]‐C(O)‐RWRWR‐NH2 and [M]‐C(O)‐WRWRW‐NH2, where [M]=[Co(Cp)(C5H4)]+, [Fe(Cp)(C5H4)] were prepared by solid‐phase peptide synthesis (SPPS). They were purified by HPLC, characterized by ESIMS and NMR spectroscopy, and tested for antibacterial properties against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus using the minimum inhibitory concentration (MIC) test. In most cases, no metal‐specific activity could be observed. However, the conjugate [Fe(Cp)(C5H4)‐C(O)‐WRWRW‐NH2] 6 was found to be particularly effective against the Gram‐positive S. aureus. The activity of this metallocene–pentapeptide conjugate (7.1 μM) was even better than the 20 amino acid naturally occurring pilosulin 2, which was used as a positive control. Unlike all other compounds tested, which were most active against the Gram‐negative E. coli strain, the ferrocene conjugate 6 was the only compound in this series that was most active against Gram‐positive bacteria. Given the health concerns resulting from multidrug resistant S. aureus strains, the incorporation of metallocenes may provide a novel line of attack.
Vapor‐Phase Beckmann Rearrangement of Oxime Molecules over H‐Faujasite Zeolite
Authors: Sirijaraensre Jakkapan , Limtrakul Jumras
Published: September 2006

The Beckmann rearrangement (BR) plays an important role in a variety of industries. The mechanism of this reaction rearrangement of oximes with different molecular sizes, specifically, the oximes of formaldehyde (H2CNOH), Z‐acetaldehyde (CH3HCNOH), E‐acetaldehyde (CH3HCNOH) and acetone (CH3)2CNOH, catalyzed by the Faujasite zeolite is investigated by both the quantum cluster and embedded cluster approaches at the B3LYP level of theory using the 6‐31G (d,p) basis set. To enhance the energetic properties, single point calculations are undertaken at MP2/6‐311G(d,p). The rearrangement step, using the bare cluster model, is the rate determining step of the entire reaction of these oxime molecules of which the energy barrier is between 50–70 kcal mol−1. The more accurate embedded cluster model, in which the effect of the zeolitic framework is included, yields as the rate determining step, the formaldehyde oxime reaction rearrangement with an energy barrier of 50.4 kcal mol−1. With the inclusion of the methyl substitution at the carbon‐end of formaldehyde oxime, the rate determining step of the reaction becomes the 1,2 H‐shift step for Z‐acetaldehyde oxime (30.5 kcal mol−1) and acetone oxime (31.2 kcal mol−1), while, in the E‐acetaldehyde oxime, the rate determining step is either the 1,2 H‐shift (26.2 kcal mol−1) or the rearrangement step (26.6 kcal mol−1). These results signify the important role that the effect of the zeolite framework plays in lowering the activation energy by stabilizing all of the ionic species in the process. It should, however, be noted that the sizeable turnover of a reaction catalyzed by the Brønsted acid site might be delayed by the quantitatively high desorption energy of the product and readsorption of the reactant at the active center.
The fabrication and evaluation of inline coupling of microdialysis with capillary electrophoresis and its application in the determination of blood glucose
Authors: Chen Hua , Yu Yanlin , Xia Zhining , Tang Shouyuan , Mu Xiaojing , Long Sha
Published: September 2006

A novel method of inline coupling of microdialysis (MD) with CE has been investigated. A polysulfone MD membrane was in situ prepared at the inlet end of the separation capillary, which endued the capillary with an additional function of clean‐up sampling and made the pretreatment of complex matrices simple. The properties of the membrane and its influence on electrophoresis were evaluated. The membrane cut off macromolecules and particles efficiently and persistently, and changing the components of membrane‐casting solution could control the molecular weight cutoff of the membrane. Using the capillary having the MD membrane in electrophoresis, column efficiency with little reduction was achieved. The mixture of chlorpheniramine (CPA) and BSA was used to verify the analytical characteristics. Only the peak of CPA appears in the electropherograms and the RSDs of migration times and peak areas of CPA were 0.8 and 4.6%, respectively. The method was applied for the determination of glucose in human blood using an electrochemical detector. Whole blood containing glucose and macromolecules was electrokinetically injected directly into the capillary without pretreatment, and the concentration of glucose in human blood was 5.5 mmol/L. Equilibrium dialysis injection is also investigated in this paper. The results show that the method may be further used for in vivo sampling to monitor the endogenous or exogenous small molecules and their metabolites.
Capillary electrochromatographic separation of proteins on a column coated with titanium dioxide nanoparticles
Authors: Hsieh Yi‐Ling , Chen Tse‐Hsien , Liu Chuen‐Ying
Published: September 2006

A TiO2 nanoparticle (TiO2 NP)‐coated open‐tubular column for the capillary electrochromatographic separation of proteins is described. The surface chemistry of the TiO2 NPs on the inner wall of the fused silica was significantly affected by the running buffer. By varying of the phosphate buffer pH, only cathodic EOF was indicated. The results showed that TiO2 NPs are existed as a complexed form with the buffer ligand. Good separation of conalbumin (ConA), apo‐transferrin (apoTf), ovalbumin (OVA), and BSA could be achieved with phosphate buffer (40 mM, pH 8.0) and an applied voltage of 15 kV. Five peaks of glycoisoforms of OVA were observed under these conditions. In comparison with the retention behavior of the analytes on the bare fused‐silica column, the new column's high resolving power seems to be predominantly derived from the ligand exchange of the analytes with the phosphate adsorbed onto the TiO2 NPs. The method was also used to separate egg‐white proteins. Both acidic and basic proteins in egg white were separated in a single run. The microheterogeneities of OVA could also be found in it. The separation efficiency for the main peak of OVA in egg white was around 10 000 plates/m.
Hybrid silica monolithic column for capillary electrochromatography with enhanced cathodic electroosmotic flow
Authors: Hu Jiwei , Xie Chuanhui , Tian Ruijun , He Zhike , Zou Hanfa
Published: September 2006

A hybrid silica monolithic stationary phase for RP CEC was prepared by in situ co‐condensation of (3‐mercaptopropyl)‐trimethoxysilane (MPTMS), phenyltriethoxysilane (PTES), and tetraethoxysilane (TEOS) via a sol–gel process. The thiol groups on the surface of the stationary phase were oxidized to sulfonic acids by peroxytrifluoroacetic acid. The introduced sulfonic acid moieties on the monoliths were characterized by a strong and relatively stable EOF in a broad pH range from 2.35 to 7.0 in CEC. Aromatic acids and neutral compounds can be simultaneously separated in this column under cathodic EOF. The CEC column exhibited a typical RP chromatographic mechanism for neutral compounds due to the introduced phenyl groups.
Stereoselective separations of chiral phosphinic acid pseudodipeptides by CEC using silica monoliths modified with an anion‐exchange‐type chiral selector
Authors: Preinerstorfer Beatrix , Lubda Dieter , Mucha Artur , Kafarski Paweł , Lindner Wolfgang , Lämmerhofer Michael
Published: September 2006

A nonaqueous CEC method for the simultaneous separation of the four stereoisomers of the N‐benzyloxycarbonyl phosphinic pseudodipeptide methyl ester benzyloxycarbonyl‐homophenylalanine Z‐hPheψ(PO2HCH2)Phe‐OCH3 as well as of the corresponding N‐2,4‐dinitrophenyl (DNP)‐derivative with free C‐terminal carboxylic group DNP‐hPheψ(PO2HCH2)Phe‐OH was developed. For this purpose, a monolithic silica capillary column modified with a cinchona alkaloid‐derived anion‐exchange‐type chiral selector, namely O‐9‐(tert‐butylcarbamoyl)quinidine (tBuCQD) was prepared. The mobile phase composition (ACN/methanol ratio, counterion type) was thoroughly optimized to end up with baseline resolution of all four stereoisomers with critical resolution of as high as about 2. The CEC method proved to be superior over the corresponding HPLC separations primarily due to significantly enhanced plate numbers (between 200 000 and 600 000 m−1 in CEC). Diastereoselectivity contributions arising from electrophoretic mobility differences of the diastereomers facilitated the separation of the later eluted diastereomeric peak pair (peaks III and IV), but had a negative influence on the selectivity of the earlier eluted diastereomeric peak pair (peaks I and II). The stereoselective CEC assay allowed the assessment of the stereoisomeric purity of the individual isomers which were obtained by preparative HPLC on a CHIRALPAK QD‐AX column that is based on the same tBuCQD selector. The present study demonstrates that there exist problems which are hard to solve by HPLC, yet can be conveniently solved by CEC. Moreover, it was intended to prove by this practical application that CEC with monolithic columns is robust enough to be used for solving real‐life problems.
Influence of dynamic soil–structure interaction on the nonlinear response and seismic reliability of multistorey systems
Authors: Bárcena Armando , Esteva Luis
Published: September 2006

A set of reinforced concrete structures with gravitational loads and mechanical properties (strength and stiffness) representative of systems designed for earthquake resistance in accordance with current criteria and methods is selected to study the influence of dynamic soil–structure interaction on seismic response, ductility demands and reliability levels. The buildings are considered located at soft soil sites in the Valley of Mexico and subjected to ground motion time histories simulated in accordance with characteristic parameters of the maximum probable earthquake likely to occur during the system's expected life. For the near‐resonance condition the effects of soil–structure interaction on the ductility demands depend mainly on radiation damping. According to the geometry of the structures studied this damping is strongly correlated with the aspect ratio, obtained by dividing the building height by its width. In this way, for structures with aspect ratio greater than 1.4 the storey and global ductility demands increase with respect to those obtained with the same structures but on rigid base, while for structures with aspect ratio less than 1.4 the ductility demands decrease with respect to those for the structures on rigid base. For the cases when the fundamental period of the structure has values very different from the dominant ground period, soil–structure interaction leads in all cases to a reduction of the ductility demands, independently of the aspect ratio. The reliability index β is obtained as a function of the base shear ratio and of the seismic intensity acting on the nonlinear systems subjected to the simulated motions. The resulting reliability functions are very similar for systems on rigid or on flexible foundation, provided that in the latter case the base rotation and the lateral displacement are removed from the total response of the system. Copyright © 2006 John Wiley & Sons, Ltd.