bluuebunny/crossref_metadata_2025 huggingface dataset:
Stereocontrolled α‐Alkylation of Fully Protected L‐Serine
Authors: Brunner Martin , Saarenketo Pauli , Straub Thomas , Rissanen Kari , Koskinen Ari M. P.
Published: August 2004

Diastereoselective alkylation of the (2S,4S) and (2R,4S) diastereomers of 3‐tert‐butyl 4‐methyl 2‐tert‐butyl‐1,3‐oxazolidine‐3,4‐dicarboxylate (1a/b) is reported. Formation of both diastereomers of these oxazolidines was achieved by changing the order of protection steps, and their relative and absolute configurations were determined by NOESY spectroscopy. The use of the bulky ring substituent tBu together with Boc as the N‐protecting group led to the exclusive formation of only one alkylated diastereomer. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Synthesis of γ‐Lactones from Cycloocta‐1,5‐diene − Starting Materials for Natural‐Product Synthesis
Authors: Behr Sandra , Hegemann Klaus , Schimanski Holger , Fröhlich Roland , Haufe Günter
Published: August 2004

The bislactones rac‐tetrahydro‐2,2′‐bifuranyl‐5,5′‐dione (rac‐12) and its diastereomer meso‐25 were prepared from endo‐5‐hydroxy‐9‐oxabicyclo[4.2.1]nonan‐2‐one (endo‐10) and endo‐6‐hydroxy‐9‐oxabicyclo[3.3.1]nonan‐2‐one (endo‐11) or exo‐5‐hydroxy‐9‐oxabicyclo[4.2.1]nonan‐2‐one (exo‐23), respectively, under the conditions of a Baeyer−Villiger oxidation with trifluoroperacetic acid. The latter compounds were obtained by O‐heterocylization of cis,cis‐cycloocta‐1,5‐diene (1) by either reaction with peracids followed by hydrolysis and Jones oxidation or ruthenium tetraoxide oxidation, respectively. The optically active bislactone (R,R)‐(−)‐12 was prepared in a similar manner from (1S,5R,6R)‐(+)‐10 and (1R,5R,6R)‐(+)‐11, which, in turn, were obtained by lipase‐catalyzed asymmetric acetylation of the corresponding diols meso‐2 and rac‐3 and subsequent Jones oxidation of the formed hydroxy esters (1S,2S,5R,6R)‐(+)‐4 and (1R,2R,5R,6R)‐(+)‐5. Since the regioisomeric hydroxy‐9‐oxabicyclo[4.2.1]‐ and ‐[3.3.1]nonan‐2‐ones (1S,5R,6R)‐(+)‐10 and (1R,5R,6R)‐(+)‐11 yielded the same bislactone [(R,R)‐(−)‐12] it is presumed that the sequence proceeds via open‐chain intermediates. Applying this strategy to the enantiopure acetoxy ketones (1S,5R,6R)‐(+)‐8 and (1R,5R,6R)‐(+)‐9, followed by Kolbe electrolysis of the formed (R,R)‐5‐acetoxy‐7‐carboxyheptan‐4‐olide [(R,R)‐27], (R,R)‐5‐acetoxydecan‐4‐olide [(R,R)‐29] was accessible in a five‐step synthesis. The absolute configuration of (+)‐8 was determined by X‐ray analysis of the dithiepane derivative 14. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
A New Carrier for Selective Removal of Heavy Metal Ions from Aqueous Solutions through Bulk Liquid Membranes
Authors: Spreti Nicoletta , Brinchi Lucia , Germani Raimondo , Mancini Maria Vincenza , Savelli Gianfranco
Published: August 2004

The carrier‐mediated transport of heavy metal ions through bulk liquid membranes has been examined: toxic Hg2+, Cd2+ and Pb2+ ions were studied, along with Cu2+ ions for comparative purposes. The ability of a new carrier, 2,2′‐bis(p‐octyloxybenzyl)diethylenetriamine (bis‐pODET), to complex and transport all the selected metal ions is reported. Differing affinities of the carrier for the different metal ions and the different experimental conditions required for their release into the receiving phase allowed the selective separation of equimolar binary mixtures. For Hg2+/Cd2+ and Hg2+/Pb2+ mixtures, two different separation methods were performed, while the inefficacy of the separation of Cd2+/Pb2+ and Hg2+/Cu2+ mixtures was for two different reasons: (i) the carrier is able to extract the metal ions with similar levels of ability, and (ii) the carrier metal ion complexes require the same acidity of the receiving phase to release the metal ions. The capability of the carrier to transport Hg2+ efficiently in consecutive cycles is also reported: over 90% of the metal ions were transferred into the receiving phase for three consecutive processes. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
General Route to a New Class of Homochiral Azetidine‐Derived 1,2‐Diamines
Authors: Couty François , Evano Gwilherm , Prim Damien , Marrot Jérome
Published: August 2004

A short access to novel azetidine‐derived anti‐1,2‐diamines starting from easily accessible 2‐cyanoazetidines has been developed. A one‐pot sequence, including a nucleophilic addition and reduction of the resulting imine, allows for a diastereoselective synthesis of variously substituted diamines. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Structural Determination of the O‐Chain Moieties of the Lipopolysaccharide Fraction from Agrobacterium radiobacter DSM 30147
Authors: De Castro Cristina , Bedini Emiliano , Garozzo Domenico , Sturiale Luisa , Parrilli Michelangelo
Published: August 2004

Two O‐chain structures were identified after acid hydrolysis of the lipopolysaccharide fraction of Agrobacterium radiobacter (type strain). The first is constituted by the linear tetrasaccharide repeating unit [2)‐α‐L‐Rhap‐(1→3)‐α‐L‐Rhap‐(1→3)‐α‐L‐Rhap‐(1→2)‐α‐L‐Rhap‐(1→]n and the second by the (1→2)‐branched repeating unit α‐D‐Manp‐(1→2)−[3)‐α‐D‐Fucp‐(1→3)‐α‐D‐Fucp‐(1→]n.The two structures were determined mainly by 1D‐ and 2D NMR spectroscopy together with chemical‐degradation methods. A detailed analysis of the NOESY spectrum, supported by molecular dynamics calculations, suggested that some unexpected NOEs were due to the sum of many small dipolar effects, whose identification was possible only by considering the 3D structure of an O‐chain oligosaccharide bigger than the repeating unit. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Glyco‐SAMs as Glycocalyx Mimetics: Synthesis of L‐Fucose‐ and D‐Mannose‐Terminated Building Blocks
Authors: Kleinert Mike , Röckendorf Niels , Lindhorst Thisbe K.
Published: August 2004

In the course of a project on the supramolecular functions of the glycocalyx present on eukaryotic cell surfaces, it has been our goal to prepare spacer glycosides and cluster glycosides that are suitable for the formation of self‐assembled monolayers (SAMs) on gold. We have selected amino‐functionalized D‐mannose and L‐fucose derivatives for peptide coupling to thio‐functionalised alkane and alkane‐oligoethylene glycol spacers such as 12 and 15. Thus, a variety of thio‐spacered glycosides (16−19) and cluster glycosides (23, 24, 26, 28, and 29) were synthesized, which can be assembled on gold wafers to serve as glycocalyx mimetics. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
A Stereoselective Formal Synthesis of (−)‐Fumagillol
Authors: Bedel Olivier , Haudrechy Arnaud , Langlois Yves
Published: August 2004

A novel formal synthesis of fumagillol, a direct precursor of the antiangiogenic sesquiterpene fumagillin, is described. The main features of the synthesis are a stereoselective Claisen−Ireland rearrangement, a ring‐closing metathesis, a chemo‐ and stereoselective dihydroxylation, and a Julia−Kocienski olefination. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Synthesis and Preparative Resolution of the trans‐Cyclohexane Analogues of Phenylalanine
Authors: Cativiela Carlos , López Pilar , Lasa Marta
Published: August 2004

Different approaches to the synthesis of enantiomerically pure (1R,2S)‐ and (1S,2R)‐1‐amino‐2‐phenylcyclohexanecarboxylic acids (trans‐c6Phe) through a racemic pathway followed by semi‐preparative HPLC of a racemic precursor have been studied. The complete diastereoselectivity of the Strecker reaction and the high efficiency of the subsequent transformations into the amino acid favour this method in comparison to the Diels−Alder route. The relative stereochemistry of the amino acid and its precursors has been unambiguously assigned. The preparation of the final enantiomerically pure amino acids and their corresponding N‐Boc derivatives was carried out by HPLC resolution of one of the intermediates using a cellulose‐derived chiral stationary phase. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Stereoselective Sensing by Substrate‐Controlled syn/anti Interconversion of a Stereodynamic Fluorosensor
Authors: Tumambac Gilbert E. , Mei Xuefeng , Wolf Christian
Published: August 2004

A stereoselective sensing method that is based on the unique stereodynamic and photochemical properties of 1,8‐diquinolylnaphthalenes has been developed. The ability to undergo substrate‐induced syn/anti interconversion and the striking difference in the fluorescence intensity of the syn and anti isomers of 1,8‐bis(2‐isopropyl‐4‐quinolyl)naphthalene (1) have been utilized for stereoselective sensing of the isomers of 1,2‐diaminocyclohexane (5). The conformational stability of 1 at room temperature greatly facilitated spectroscopic and crystallographic studies of this new prototype of stereodynamic fluorosensors. Fluorescence measurements of the anti and syn isomer of 1 revealed a quantum yield of 11.6% and 2%, respectively. NMR spectroscopy experiments confirmed that trans‐5 stabilizes the C2‐symmetric anti isomers of 1, whereas cis‐5 favors formation of the less fluorescent meso syn‐1 isomer, which results in a reduced fluorescence response. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Ciereszkolide: Isolation and Structure Characterization of a Novel Rearranged Cembrane from the Caribbean Sea Plume Pseudopterogorgia kallos
Authors: Marrero Jeffrey , Rodríguez Abimael D. , Baran Peter , Raptis Raphael G.
Published: August 2004

An investigation of the chloroform extract of a Colombian specimen of Pseudopterogorgia kallos has led to the isolation of a novel rearranged cembrane, ciereszkolide (1). The structure of 1 is based on a new carbon skeleton that possesses several unusual structural features, namely, two butyrolactones [one 2(3H)‐ and one 2(5H)‐furanone] across a 13‐membered macrocycle, a (Z)‐trisubstituted olefin, and a five‐membered trans‐fused cyclic ketal. The structure of 1 was resolved by interpretation of 1D and 2D NMR spectroscopic data supported by HRFAB‐MS, IR, UV, and a single‐crystal X‐ray diffraction analysis. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Convenient Synthesis, Characterization and GPx‐Like Catalytic Activity of Novel Ebselen Derivatives
Authors: Zade Sanjio S. , Panda Snigdha , Tripathi Santosh K. , Singh Harkesh B. , Wolmershäuser Gotthelf
Published: August 2004

The synthesis and characterization of benzisochalcogenazolones (ebselen derivatives 16−21) is described. The synthesis of 16−21 was achieved by treating the bromo precursors 11−13 with an appropriate dilithium dichalcogenide. The synthesis of benzisoselenazolones 16 and 18 was also accomplished by an alternative route, that is, by treating the corresponding methoxymethyl selenides 22 and 23 with 1 equiv. of bromine. The synthesis of methoxymethyl selenides 22 and 23 was accomplished by lithiation of the bromo precursors followed by treatment with bis(methoxymethyl) diselenide. The benzisoselenazolones 16 and 17 were characterized by single‐crystal X‐ray techniques. The GPx‐like catalytic activities of compounds 16−18 and 21 were determined by using the coupled reductase assay. Compound 16 was found to be less active than ebselen 8 whereas compounds 17 and 18 were more active than ebselen in this assay. Compound 21 showed a 1.5‐fold higher activity than its selenium analogue 17. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
The “Azirine/Oxazolone Method” under Solid‐Phase Conditions
Authors: Stamm Simon , Heimgartner Heinz
Published: August 2004

Aib‐containing peptides have been synthesized from the N‐ to the C‐terminus by the “azirine/oxazolone method” under solid‐phase conditions. In this new and convenient method for the synthesis of sterically demanding peptides on solid phase, 2H‐azirine‐3‐amines are used to introduce aminoisobutyric acid, an α,α‐disubstituted α‐amino acid, into the peptide without the need for further reagents. Segments of naturally occurring peptaibols have been prepared by this method. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Synthesis and Evaluation of Boronated Lysine and Bis(carboranylated) γ‐Amino Acids as Monomers for Peptide Assembly
Authors: Morin Christophe , Thimon Christian
Published: August 2004

We have synthesizedN‐Fmoc amino acid derivatives bearing boronated and carboranylated substituents. Thus, a 1,3,2‐dioxaborolane subunit has been introduced on the side chain ofNα‐Fmoc‐L‐lysine, and a bis(carboranylated) γ‐amino acid building block has been prepared. As shown by the preparation of a model tetrapeptide, only the latter monomer can be successfully used in peptide synthesis. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Tandem Intramolecular Carbolithiation‐Lithium/Zinc Transmetallation and Applications to Carbon−Carbon Bond‐Forming Reactions
Authors: Yus Miguel , Ortiz Rosa
Published: August 2004

Lithium/zinc transmetallation with the cyclic organolithium intermediate 3 (prepared by intramolecular carbolithiation of the initially formed organolithium 2) gives the corresponding organozinc intermediate 5. Copper‐ or palladium‐promoted SN2′ reactions between compound 5 and allylic or propargylic halides, as well as arylation, vinylation, benzylation, acylation, and conjugate addition to α,β‐unsaturated carbonyl compounds, afford (after hydrolysis) the expected compounds 7−15, which are not accessible directly from the organolithium precursor 3 even with transition metal catalysis. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Copper‐Catalyzed Synthesis of N‐Unsubstituted 1,2,3‐Triazoles from Nonactivated Terminal Alkynes
Authors: Jin Tienan , Kamijo Shin , Yamamoto Yoshinori
Published: August 2004

The [3+2] cycloaddition of nonactivated terminal alkynes and trimethylsilyl azide proceeded smoothly in the presence of CuI catalyst and DMF/MeOH, to give the corresponding N‐unsubstituted triazoles in good to high yields. The reaction most probably proceeds through the in situ formation of a copper acetylide species and hydrazoic acid, followed by a successive [3+2] cycloaddition reaction. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)
Cover Picture: Molecular “Iron Maidens”: Ultrashort Nonbonded Contacts in Cyclophanes and Other Crowded Molecules (Eur. J. Org. Chem. 17/2004)
Authors: Pascal, Robert A.
Published: August 2004

The cover picture shows the last step in the synthesis of an in‐cyclophane. Like the medieval “iron maiden”, an instrument of torture in which the closing of a spiked door impaled a victim, the final ring closure forces a functional group into the face of a benzene ring. Details are discussed in the Microreview by R. A. Pascal, Jr. on p. 3763 ff.
Disposable twin gold electrodes for amperometric detection in capillary electrophoresis
Authors: Richter Eduardo M. , Fracassi da Silva José A. , Gutz Ivano G. Rolf , do Lago Claudimir L. , Angnes Lúcio
Published: August 2004

We describe a simple and easy way to construct gold microelectrodes for amperometric detection in capillary electrophoresis (CE). The gold microelectrodes, in single or twin sets, were obtained from recordable compact discs (gold‐sputtered type), which present highly reproducible surface characteristics. The performance of these electrodes was evaluated by using a home‐made CE equipment. The basic steps for the electrode construction are: drawing on a microcomputer; laser printing of the design on wax paper; heat‐transfer of the toner onto the gold surface of a peeled recordable compact disc (CD‐R); etching of the gold layer from unprinted regions; removal of the toner with a solvent; sealing of unused electrode areas with varnish. One electrode at a time was connected to a potentiostat (or two, to a bipotentiostat) and operated in a wall jet configuration relative to the CE capillary outlet. The amperometric signals were integrated for quantification purposes. Repetitive injections (n = 10) of a mixture containing iodide, ascorbic acid, dipyrone, and acetaminophen (20, 200, 500, and 100 μM), presented relative standard deviations of 2.9, 4.5, 6.1, and 4.0%, respectively. For these analytes, the detection limits (S/N = 3, 30 s of 100 mm hydrodynamic injection) were 0.1, 0.5, 3.1, and 1.1 μM, respectively.
Microchip capillary electrophoresis with a boron‐doped diamond electrochemical detector for analysis of aromatic amines
Authors: Shin Dongchan , Tryk Donald A. , Fujishima Akira , Muck Alexander , Chen Gang , Wang Joseph
Published: August 2004

The attractive features of a boron‐doped diamond (BDD) thin‐film detector for microchip capillary electrophoretic (CE) separations of dye‐related amino‐substituted aromatic compounds are described. The diamond electrode was employed in the end‐column amperometric detection of 4‐aminophenol (4‐AP), 1,2‐phenylenediamine (1,2‐PDA), 2‐aminonaphthalene (2‐AN), 2‐chloroaniline (2‐CA), and o‐aminobenzoic acid (o‐ABA), and its attractive behavior was compared to commonly used screen‐printed carbon and glassy‐carbon electrodes. These conventional electrode materials exhibit a significant degree of passivation and low sensitivity to the above‐mentioned environmental pollutants. The diamond‐based electrochemical detection system displayed a favorable analytical performance, including lower noise levels, higher peak resolution with enhanced sensitivity, and improved resistance against electrode passivation. Factors influencing the on‐chip analysis were assessed and optimized. The diamond detector displayed detection limits of 2.0 and 1.3 μM for 4‐AP and 2‐AN, respectively, and a wide linear response for these compounds over the 2–50 μM range. The enhanced stability was demonstrated by relative standard deviation (RSD) values of 1.4% and 4.7% for 100 μM 1,2‐PDA and 200 μM 2‐CA, respectively, for repetitive detections (n = 7). Besides, the simultaneously observed current decrease was 2.4 and 9.1% for 1,2‐PDA and 2‐CA, respectively (compared to 21.8 and 41.0% at the screen‐printed carbon electrode and 28.3 and 34.1% at the glassy carbon electrode, respectively). The favorable properties of the diamond electrode indicate great promise for environmental applications in CE and other microchip devices.
Determination of SARS‐coronavirus by a microfluidic chip system
Authors: Zhou Xiaomian , Liu Dayu , Zhong Runtao , Dai Zhongpeng , Wu Dapeng , Wang Hui , Du Yuguang , Xia Zhinan , Zhang Liping , Mei Xiaodai , Lin Bingcheng
Published: August 2004

We have developed a new experimental system based on a microfluidic chip to determine severe acute respiratory syndrome coronavirus (SARS‐CoV). The system includes a laser‐induced fluorescence microfluidic chip analyzer, a glass microchip for both polymerase chain reaction (PCR) and capillary electrophoresis, a chip thermal cycler based on dual Peltier thermoelectric elements, a reverse transcription‐polymerase chain reaction (RT‐PCR) SARS diagnostic kit, and a DNA electrophoretic sizing kit. The system allows efficient cDNA amplification of SARS‐CoV followed by electrophoretic sizing and detection on the same chip. To enhance the reliability of RT‐PCR on SARS‐CoV detection, duplex PCR was developed on the microchip. The assay was carried out on a home‐made microfluidic chip system. The positive and the negative control were cDNA fragments of SARS‐CoV and parainfluenza virus, respectively. The test results showed that 17 positive samples were obtained among 18 samples of nasopharyngeal swabs from clinically diagnosed SARS patients. However, 12 positive results from the same 18 samples were obtained by the conventional RT‐PCR with agarose gel electrophoresis detection. The SARS virus species can be analyzed with high positive rate and rapidity on the microfluidic chip system.
Determination of pore/protein size via electrophoresis and slit sieve model
Authors: Sarbolouki Mohammad N. , Mahnam Karim , Rafiee‐Pour Hossain‐Ali
Published: August 2004

We used electrophoresis for three purposes: (i) estimation of the mean pore size of polyacrylamide gels via measuring electrophoretic mobility of globular proteins of known sizes in combination with simple sieve (cylindrical and slit) models; (ii) determination of the average size of protein molecules (native or denatured) by the use of the same models; (iii) monitoring the changes in molecular dimensions of proteins in the course of their denaturation. Both models yield results that are in good agreement with those found via the more elaborate techniques (considering the principal differences involved). The approach provides a direct and convenient way of monitoring the variations in protein sizes during the course of their denaturation in gels having a gradient of denaturants, and possibly the number of conformational states involved in the process, a facet that is quite unique and useful. The simpler slit model seems to yield better results in the latter case and is moreover supported by the recently reported data on electrophoresis of DNA molecules through the 1 μm slits of a microbrush matrix made of micropillars arranged in a hexagonal lattice.
Capillary isoelectric focusing – reproducibility and protein adsorption
Authors: Graf Michael , Wätzig Hermann
Published: August 2004

Capillary isoelectric focusing (CIEF) is an important tool for the quality assurance of biotechnologically maintained drugs and for proteome analysis. The critical performance parameters of this technique are the precisions of isoelectric point (pI) values and peak areas. Compared to capillary zone electrophoresis (CZE), where precise results can be obtained (e.g., 0.5% relative standard deviation (RSD) for peak areas, n = 60), only few data are available for CIEF experiments. So far, reproducible data of pI values (RSD = 0.5%) have been acquired, but peak areas show inferior results (about 3–15% RSD). Nonstable capillary coatings and protein adsorption have been discussed as possible reasons. Recent work of Righetti et al. [25, 27] has proven that the use of coated capillaries can reduce the adsorption of proteins by 50% but cannot prevent it. In our CIEF experiments irregular and poorly reproducible peak patterns have been observed. In a long‐time experiment of 106 repeated runs, an overall RSD of 10% was obtained for peak areas, RSD of 2% only in series of about 10 consecutive replicates. Especially at higher concentrations the reproducibility deteriorates. This seems to be the result of a self‐amplifying process, induced by adsorbed protein molecules, leading to further agglomerations. CZE control experiments in linear polyacrylamide (LPA)‐coated capillaries proved a strong pH dependency of these effects within a small range. Compared to bare fused‐silica surfaces, adsorption effects are reduced but not inhibited. An enhancement of reproducibility in CIEF experiments can be achieved only by controlling the interactions of proteins and capillary walls.
Separation and determination of aconitine alkaloids in traditional Chinese herbs by nonaqueous capillary electrophoresis
Authors: Li Yuqin , Qi Shengda , Chen Xingguo , Hu Zhide
Published: August 2004

An easy, rapid, and simple nonaqueous capillary electrophoresis (NACE) method was developed for the identification and determination of three aconitine alkaloids, hypaconitine (HN), aconitine (AN), and mesaconitine (MN) within 6 min. The most suitable running buffer was composed of 60 mM ammonium acetate, 0.5% acetic acid, and 15% acetonitrile (ACN) in methanol with a fused‐silica capillary column (50 cm×75 μm ID). In the concentration range 12.5–1000 mg/L the calibration curves reveal linear relationships between the peak area for each analyte and its concentration (correlation coefficients: 0.9997 for HN, 0.9999 for AN, and 0.9995 for MN). The relative standard deviations of the migration time and peak area of the three alkaloids were 0.13, 0.57, 0.33 and 2.87, 1.06, 3.49%, respectively. The method was successfully applied to determine the three alkaloids in two commonly used traditional Chinese herbal medicines, the recoveries of the three constituents ranging between 94.7–101.9% for HN, 98.3–102.3% for AN, and 98.1–104.6% for MN.
Cerebrospinal fluid amyloid β peptide patterns in Alzheimer's disease patients and nondemented controls depend on sample pretreatment: Indication of carrier‐mediated epitope masking of amyloid β peptides
Authors: Bibl Mirko , Esselmann Hermann , Otto Markus , Lewczuk Piotr , Cepek Lukas , Rüther Eckart , Kornhuber Johannes , Wiltfang Jens
Published: August 2004

A quantitative urea‐based amyloid β (Aβ)‐sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with Western immunoblot (Aβ‐SDS‐PAGE/immunoblot) reveals highly conserved and disease‐specific Aβ peptide patterns (Aβ 1‐37, 1‐38, 1‐39, 1‐40, 1‐42) in Alzheimer's disease (AD) patients and nondemented controls. For further standardization of this method, we analyzed cerebrospinal fluid (CSF) of eight probable AD patients and seven nondemented controls using different preanalytical procedures for Aβ‐SDS‐PAGE/immunoblot and Aβ1‐42‐enzyme linked immunosorbent assay (ELISA). Both diagnostic groups were discriminated significantly by absolute levels of Aβ1‐42 and ratios of Aβ1‐42/40, 1‐42/38, 1‐42/39. Preanalytical freezing of CSF led to a highly significant loss of all Aβ peptide species. This effect was most pronounced for Aβ1‐42 and completely prevented by SDS‐heat denaturation prior to freezing. Prolonged storage of SDS‐heat denatured CSF led to a selective loss of Aβ1‐42 and impaired the discrimination of diagnostic groups as measured by Aβ‐SDS‐PAGE/immunoblot. Neither freezing nor storage significantly affected absolute Aβ1‐42 levels as determined by Aβ1‐42‐ELISA, but both impaired the discrimation of diagnostic groups. Hence, we suggest immediate analysis of samples for Aβ1‐42‐ELISA, analysis after a short freezing interval for Aβ‐SDS‐PAGE/immunoblot, and avoidance of prolonged storage intervals. Remarkably, Aβ‐SDS‐PAGE/immunoblot measured threefold higher levels of Aβ1‐42 in CSF than Aβ1‐42‐ELISA. In summary, our results indicate carrier‐mediated epitope masking of Aβ1‐42.
Separation and recovery of intact gold‐virus complex by agarose electrophoresis and electroelution: Application to the purification of cowpea mosaic virus and colloidal gold complex
Authors: Soto Carissa M. , Szuchmacher Blum Amy , Wilson Charmaine D. , Lazorcik Jason , Kim Moon , Gnade Bruce , Ratna Banahalli R.
Published: August 2004

Colloidal gold has been coupled to a mutant cowpea mosaic virus (CPMV), which contains 60 cysteine residues on the surface. A purification process was developed to separate the gold‐containing viral nanoblocks (VNBs) from the free gold. Agarose electrophoresis was utilized to separate the mixture followed by electroelution of the desired sample to recover the intact virus. Mobility of Au‐VNB and free colloidal gold was facilitated by the addition of thioctic acid (TA). 30% of the gold‐containing virus was recovered after electroelution as determined by absorbance measurements. Histogram analysis of transmission electron microscopy (TEM) images demonstrated the efficient separation of gold‐containing virus from free gold. TEM and scanning electron microscopy (SEM) images indicated that the virus was recovered intact. Monodisperse spherical particles of nominal size of 45 nm were observed under SEM.
Determination of amino acids in rat vitreous perfusates by capillary electrophoresis
Authors: Thongkhao‐On Kongthong , Kottegoda Sumith , Pulido Jose S. , Shippy Scott A.
Published: August 2004

In vivo determinations of amino acids are important for improving our understanding of physiological states of biological tissue function and dysfunction. However, the chemically complex matrix of different biological fluids complicates the assay of this important class of molecules. We introduce a method for characterizing the amino acid composition of submicroliter volumes of vitreous humor perfusates. Low‐flow push‐pull perfusion sampling is compatible with collecting small volume samples in a complicated matrix that are potentially difficult to separate. An efficient, sensitive, and rapid analysis of amino acids from in vivo perfusates of the vitreous is presented with 3‐(4‐carboxybenzoyl)‐2‐quinoline‐carboxaldehyde (CBQCA) derivatitation and capillary electrophoresis (CE) separation with laser‐induced fluorescence detection (LIF). Derivatization with CBQCA for up to 2 h provided high sensitivity and low detection limits at the nM level. Seventeen amino acids including D‐serine (D‐Ser) and D‐aspartate (D‐Asp) were resolved in less than 10 min. Importantly, D‐Ser is separated from its enantiomeric pair. Characterization of vitreal amino acids with this assay technique will be useful for understanding ocular diseases and physiological mechanisms in vision.
A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis
Authors: White Ian R. , Pickford Russell , Wood John , Skehel J. Mark , Gangadharan Bevan , Cutler Paul
Published: August 2004

Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two‐dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex‐based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.
High reproducibility of large‐gel two‐dimensional electrophoresis
Authors: Challapalli Kiran Kumar , Zabel Claus , Schuchhardt Johannes , Kaindl Angela M. , Klose Joachim , Herzel Hanspeter
Published: August 2004

Two‐dimensional gel electrophoresis (2‐DE) facilitates the separation of thousands of proteins from highly complex protein mixtures and has become a central method in proteomics in recent years. In the present study, we examined the technical variability of large 2‐DE gels with respect to sample preparation, electrophoresis procedure, data acquisition, and biological variation by analyzing a disease (Huntington's disease) and control state with a commercially available software package, PROTEOMWEAVER™. Scatter plots and correlation coefficients were obtained to quantify both technical and biological variation. Even 2‐DE gels run separately in both dimensions yielded correlation coefficients around 0.88 and deviations from the mean close to 20% for low‐intensity spots. This indicates a high technical reproducibility of the 2‐DE procedure developed in our laboratory. Variability within a biological condition was low and comparable to technical variation (at least 0.87). Two‐dimensional (2‐D) gels obtained from samples of different biological conditions (health vs. disease) achieved a variability similar to intracondition and technical variability. These findings highlight the importance of multiple gel and spot‐by‐spot comparisons to identify biological significant changes. Minor errors introduced by technical and biological variation allow a comparison of all gels within a study which facilitates the tackling of complex biological problems.
Separation of proteins on surface‐modified poly(dimethylsiloxane) microfluidic devices
Authors: Dou Yue‐Hua , Bao Ning , Xu Jing‐Juan , Meng Fei , Chen Hong‐Yuan
Published: August 2004

Separation and detection of proteins have been realized on nonionic surfactant‐modified poly(dimethylsiloxane) (PDMS) microfabricated devices with end‐column amperometric detection. The hydrophobic PDMS channels are turned into hydrophilic ones after being modified with Brij35 and facilitate the separation of proteins. The coating can remarkably reduce the adsorption of large protein molecules and is stable in the range of pH 6–12. The detection of proteins in such channels needs less rinsing time and thus efficiency is raised. Even large molecules of proteins can also be detected with better reproducibility and enhanced plate numbers. The relative standard deviation (RSD) of the migration time for glucose oxidase (GOD) is 2.2% (n = 19). Separation of GOD and myoglobin has been developed in modified channels. Predominant operational variables, such as the coating conditions, the concentration of surfactant and buffer, are studied in detail.
Capillary and microchip electrophoresis of basic drugs with contactless conductivity detection
Authors: Tanyanyiwa Jatisai , Hauser Peter C.
Published: August 2004

The extension of contactless conductivity detection in electrophoresis to the determination of basic drugs is demonstrated using β‐adrenergic blocking agents (β‐blockers) and other physiologically active amines as examples. The high‐voltage approach to conductivity detection was employed for conventional capillaries as well as microchip devices. Acidic buffers were used in all cases. A buffer consisting of 100 mM acetic acid and 1 mM histidine was deemed most optimal for the separation of six β‐blockers and best results for the analysis of the other amines were achieved with a 20 mM lactic acid buffer at low pH‐value. The detection limits ranged from 0.06 to 5 μM. To demonstrate potential practical applications, a main component assay was conducted for three pharmaceutical formulations. On‐chip, five pharmaceutical amines could be baseline‐resolved in a 8 cm long microchannel in 90 s, albeit a reduced sensitivity and peak capacity compared to conventional capillary electrophoresis.
Nonlinear electroosmosis in hierarchical monolithic structures
Authors: Nischang Ivo , Tallarek Ulrich
Published: August 2004

We studied the dependence of electroosmotic flow (EOF) velocity and separation efficiency for neutral analytes in 100 μm ID capillary monoliths on a variation of the mobile phase ionic strength and applied electrical field strength, i.e., we covered a range for the concentration of Tris buffer from 10−5 to 10−2 M and applied electrical field strengths up to 105 V/m. The silica‐based monoliths are hierarchically structured having intraskeleton mesopores and interskeleton macropores. While a linear dependence of the average EOF velocity on applied field strength could be observed with 5×10−3 M Tris (turning slightly nonlinear at a higher concentration due to thermal effects), this dependence becomes systematically nonlinear as the Tris concentration is reduced towards 10−4 M. Increased velocities by more than 50% compared to those expected from linear behavior are realized at 105 V/m. Concomitantly, as the Tris concentration is reduced from 10−3 to 10−4 M, we notice an improvement in plate heights by a factor of more than 2 (they approach 2 μm for ethylbenzoate). We complementary analyzed the onset of the nonlinear EOF dynamics in a hierarchical monolith and the significantly reduced axial dispersion in view of nonequilibrium electrokinetic effects which may develop in porous media due to the presence of ion‐permselective regions, e.g., the mesoporous monolith skeleton. In this respect, a decreasing mobile phase ionic strength favors the formation of nonequilibrium concentration polarization in strong electrical fields, and a coupling of the electrostatics and hydrodynamics then may explain nonlinear EOF velocities and increasing separation efficiencies depending on the Tris concentration and applied field strength.
In situ monitoring of kinetics of metabolic conversion of ATP to ADP catalyzed by MgATPases of muscle Gastrocnemius skinned fibers using micellar electrokinetic chromatography
Authors: Kulp Maria , Kaljurand Mihkel , Käämbre Tuuli , Sikk Peeter , Saks Valdur
Published: August 2004

A method for the in situ measurement of the kinetics of ATP metabolic transformation using capillary electrophoresis (CE) has been developed. The depletion of ATP and formation of ADP were monitored in situ by using saponin‐permeabilized muscle fibers. The method of micellar electrokinetic chromatography, employing reversed electroosmotic flow by cationic surfactant and reversed‐polarity mode, provided an efficient and reproducible separation of nucleotides and enabled kinetic analysis of the reaction to be performed in a large range of nucleotide concentrations that approaches physiological concentrations of ATP in the muscle cells, without the need for precipitation of proteins prior to sample application. The analytes were detected at a nM level with a reproducibility of about 7%. This reproducibility enabled the comparison of different competing kinetic models of ATP conversion to ADP and the results show that the MgATPase activity in the fast‐twitch gastrocnemius muscle followed biphasic kinetics that corresponds to the allosteric character of regulation of the enzyme(s) activity at physiological ATP concentrations. The results also confirmed that the combination of minimal sample volume requirements, rapid measurement and reproducibility makes the micellar CE a valuable tool for the analysis of biological fluids and understanding the processes of biological interest.
Improved resolution power of electrophoretic fractionation of DNA using a voltage gradient “up and down” application
Authors: Bellavia Daniele , Sanfilippo Rosalia , Sisino Giorgia , Palmeri Laura , Barbieri Rainer
Published: August 2004

The improved resolution power of electrophoretic fractionation of DNA in a wide range of molecular masses is demonstrated using an “up and down” application of voltage gradient gel electrophoresis (VGGE). This application also allows separation of different DNA fragments which are poorly fractionated in conventional electrophoresis.
Polyacrylamide gel electrophoresis of fluorophore‐labeled hyaluronan and chondroitin sulfate disaccharides: Application to the analysis in cells and tissues
Authors: Karousou Evgenia G. , Militsopoulou Maria , Porta Giovanni , De Luca Giancarlo , Hascall Vincent C. , Passi Alberto
Published: August 2004

This report describes a new formulation of polyacrylamide gel electrophoresis of fluorophore‐labeled saccharides (PAGEFS) for the analysis of hyaluronan (HA) and chondroitin sulfate (CS) Δ‐disaccharides. PAGEFS relies on derivatization of reducing ends of HA‐ and the variously sulfated CS‐derived Δ‐disaccharides with 2‐aminoacridone (AMAC), followed by electrophoresis under optimized buffer conditions (Tris‐borate and Tris‐HCl) and on polyacrylamide gels (25% T/3.75% C). The method was applied to the analysis of glycosaminoglycans (GAGs) from the human umbilical cord tissue and GAGs isolated from human aortic smooth muscle cell cultures. The obtained results were in agreement with those obtained after an analysis with high‐performance liquid chromatography (HPLC). On the basis of these results, PAGEFS is a rapid and sensitive method for the analysis of the total amount of HA‐ and CS‐derived disaccharides, as it allows analyzing 20 samples in minigels in one run and provides quantitation with relatively high sensitivity (less than 25 pmol per disaccharide). In addition, PAGEFS overcomes the lack of commercial gels described previously for the separation of AMAC‐labeled disaccharides. Therefore, the method proposed here is an economic and useful tool for a fast screening of GAGs in biological samples, particularly when a high number of samples should be analyzed.
A mouse serum two‐dimensional gel map: Application to profiling burn injury and infection
Authors: Duan Xunbao , Yarmush David M. , Berthiaume François , Jayaraman Arul , Yarmush Martin L.
Published: August 2004

With the importance of mouse as a model to study human diseases and the human and rat plasma/serum two‐dimensional (2‐D) maps being extensively annotated, this study was aimed at constructing a detailed mouse serum 2‐D map. Serum proteins from two different inbred strains of mice (BALB/cJ and C57BL/6J) and mice subjected to two different inflammatory stimuli (20% burn injury and lipopolysaccharide (LPS) injection) were separated on overlapping gels covering pH 3–8 and stained with SYPRO Ruby dye. The tryptic peptides from the resolved spots were analyzed by mass spectrometry, leading to the identification of 38 different gene products. With the exception of major urinary proteins found in abundance in male C57BL/6J mice, little strain difference of the mouse serum 2‐D was observed. Many proteins detected in the mouse serum 2‐D map were not reported in human or rat serum 2‐D maps including epidermal growth factor receptor. Three major murine acute‐phase proteins (APPs), haptoglobin, serum amyloid A, and serum amyloid P, were highly induced by both inflammatory stimuli. Image analysis shows that the variations of APPs between these two inflammatory models were not uniform although LPS (100 μg/animal) in general was more effective than 20% burn injury in inducing APPs. Serum amyloid A, much more sensitive to endotoxin than burn injury, may represent a sensitive marker to differentiate these two different inflammatory states.
Formamide as solvent for capillary zone electrophoresis
Authors: Porras Simo P. , Kenndler Ernst
Published: August 2004

A comprehensive investigation of a number of aspects when using formamide as background electrolyte solvent in capillary zone electrophoresis was presented. It included (i) the change of the ion mobility with ionic strength, (ii) the influence of the ionic strength on diffusion coefficients, and (iii) on the separation efficiency expressed by the maximum reachable plate numbers (when only longitudinal diffusion contributed to zone broadening), (iv) the effect of the solvent on pKa values (taken from the literature) of neutral and cation acids, (v) the establishment of the a pH scale in formamide by dissolving acids with known pKa values and their salts at defined proportion (thus circumventing the problem of calibrating the pH meter), (vi) the agreement between the experimentally derived and the theoretical dependence of the effective mobility on pH, (vii) the uptake of water of this hygroscopic solvent from the humidity of the environment and its consequence to the ion mobilities, pKa values, and the chemical stability of the solvent (e.g., hydrolysis), and finally (viii) the use of conductivity and indirect UV absorption to enable detection of analytes below the optical cutoff of formamide.
Detection of glutathione reductase after electrophoresis on native or sodium dodecyl sulfate polyacrylamide gels
Authors: Hou Wen‐Chi , Liang Hong‐Jen , Wang Ching‐Chiung , Liu Der‐Zen
Published: August 2004

Commercial glutathione reductase (GR) from spinach and yeast (Saccharomyces cerevisiae) were stained on 7.5% native polyacrylamide gel electrophoresis (PAGE) gels or 15% sodium dodecyl sulfate (SDS)‐PAGE gels with or without further purification by a 2',5'‐ADP Sepharose 4B affinity column. For SDS‐PAGE gels, the SDS was removed first by washing twice with 25% isopropanol in 10 mM Tris‐HCl (pH 7.9) for 10 min. The gel was then dipped in a 50 mM Tris‐HCl buffer (pH 7.9) containing 4.0 mM oxidized glutathione (GSSG), 1.5 mM NADPH, and 2 mM 5,5'‐dithiobis(2‐nitrobenzoic acid) (DTNB) for 20 min. The GR activity was negatively stained in the dark by a solution containing 1.2 mM 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and 1.6 mM phenazine methosulfate (PMS) for 5–10 min. The contrast between the clear zone of GR activity and the purple background was found in both native and SDS‐PAGE gels. This negative staining method can detect GR as little as 0.064 units and 0.0032 units, respectively, for spinach and yeast sources. Under reduced SDS‐PAGE gels, the GR activity band located on 72 kDa for spinach and 51 kDa for yeast. This fast and sensitive method could be used during enzyme purification and for characterization of GR from different sources under different physiological stages or conditions.
Prediction of wheat dough W and P/L inflation test parameters by capillary zone electrophoresis of a protein extract followed by multivariate regression
Authors: Sebastiano Roberto , Simó‐Alfonso Ernesto F. , Citterio Attilio , Ramis‐Ramos Guillermo
Published: August 2004

A procedure for the evaluation of the wheat flour hardness, based on capillary electrophoresis of a protein extract in an isoelectric acidic buffer, was developed. The 13 flour samples were extracted twice, and two injections of each extract were made. Separations were performed in a background electrolyte (BGE) containing 40 mM aspartic acid, 6 M urea, and 0.5% hydroxyethylcellulose at 60°C. Using the normalized and corrected areas of 79 peaks and peak groups, a partial least squares regression (PLS1) model was able to predict the flour strength or dough deformation work (W) and the dough tenacity/ extensibility ratio (P/L) (Alveograph parameters) with an average relative standard deviation in the predictions of ± 3% and ±8%, respectively. These values amounted to a ± 6–8% and ± 11% with multiple linear regression (MLR) and PLS1 models constructed by measuring only 12 peaks and peak group areas on the electropherograms.
Charge density profiling of circulating human low‐density lipoprotein particles by capillary zone electrophoresis
Authors: Liu Mine‐Yine , McNeal Catherine J. , Macfarlane Ronald D.
Published: August 2004

Capillary zone electrophoresis (CZE) has been utilized to profile the low‐density (LDL) particles in human blood serum in this study. A 5 mM sodium phosphate buffer, pH 7.40, was chosen as the most suitable CE buffer and an extensive ultrafiltration (UF) procedure was applied to purify the LDL sample. Two LDL particle species, LDL with lower mobility and LDL− with higher mobility were observed. The electropherograms were highly reproducible with good precision of effective mobilities, corrected peak areas (CPAs) and CPA ratio of LDL−/LDL. LDL particles shown on the electropherogram were also characterized by several procedures. The applications of Sigma HDL cholesterol reagent and CE on‐line 2‐propanol precipitation indicated that the two particle species shown in the electropherogram belong to LDL. The LDL particles were found to associate with the buoyant LDL fraction and the LDL− particles associate with the dense LDL fraction. This study utilizes CZE for the profiling of LDL isoforms and provides a new analytical method for the resolution of LDL subspecies. It demonstrates a high‐mobility LDL particle which circulates in healthy subjects and diminishes in atherosclerotic patients. Diminution of the high‐mobility LDL subspecies may be linked to minimal formation of arterial plaque in atherosclerotic patients.