Writing with DNA and protein using a nanopipet for controlled delivery

We present a new, general method for the controlled deposition of biological molecules on surfaces, based on a nanopipet operating in ionic solution. The potential applied to the pipet tip controls the flux of biological molecules from the pipet, allowing fine control of the delivery rate. We used the ion current to control the distance of the pipet from the surface of a glass slide and deposited the fluorescently labeled DNA or protein G at a defined location onto the surface. Features of 830 nm size were obtained by depositing the biotinylated DNA onto a streptavidin surface; 1.3 mum size spots were obtained by depositing protein G onto a positively charged glass surface.     

Investigation of the enzymatic and nonenzymatic cope rearrangement of carbaprephenate to carbachorismate

The dimethyl esters of carbaprephenate and 4-epi-carbaprephenate were prepared by modification of published procedures. In methanol these compounds are converted quantitatively to isomeric 6-hydroxytricyclo[3.3.1.0(2,7)]non-3-en-1,3-dimethyl esters via a two-step sequence involving an initial Cope rearrangement, followed by intramolecular Diels-Alder reaction of the dimethyl carbachorismate or 4-epi-carbachorismate intermediates. Carbaprephenate and its epimer were obtained by alkaline hydrolysis of the corresponding dimethyl esters. These compounds, in contrast to their ester precursors, undergo spontaneous acid-catalyzed decarboxylation in aqueous solution. Only at high pH does the Cope rearrangement compete with decarboxylation. At pH 12 and 90 degrees C, carbaprephenate slowly rearranges to carbachorismate, which rapidly loses water to give 3-(2-carboxyallyl)benzoic acid as the major product. A small amount of the intramolecular Diels-Alder adduct derived from carbachorismate is also observed by NMR as a minor product. Carbaprephenate is not a substrate for the enzyme chorismate mutase from Bacillus subtilis (BsCM), nor does carbaprephenate inhibit the normal chorismate mutase activity of this enzyme, even when present in 200-fold excess over chorismate. Its low affinity for the enzyme-active site is presumably a consequence of placing a methylene group rather than an oxygen atom proximal to the essential cationic residue Arg90. Nevertheless, BsCM variants that lack this cation (R90G and R90A) do not accelerate the Cope rearrangement of carbaprephenate either, and a catalytic antibody 1F7, which exhibits modest chorismate mutase activity, is similarly inactive. Poor substrate binding and the relatively high barrier for the Cope compared to the Claisen rearrangement presumably account for the lack of detectable catalysis. Acceleration of this sigmatropic rearrangement apparently requires more than an active site that is complementary in shape to the reactive substrate conformer.     

Impact of adsorption isotherm parameters on the performance of enantioseparation using simulated moving bed chromatography

Often there are several chromatographic systems, i.e., combinations of mobile and stationary phases, available to solve a certain separation problem. Essential differences of these chromatographic systems are the separation factors and the efficiencies. For preparative applications in addition also the column saturation capacities and solubility limits are of importance. The impact of all these parameters appears to be rather well understood for conventional overloaded elution chromatography using a single column. In the last years the continuous simulated moving bed (SMB) process was increasingly used as a powerful alternative to batch elution since increased productivities and reduced solvent consumptions could be realised. However, the selection of suitable chromatographic systems is more sophisticated for this process. In this paper five different chromatographic systems capable of separating the enantiomers of mandelic acid are compared based on the achievable productivities using SMB chromatography. For these five systems the adsorption isotherms have been determined experimentally. Subsequently, an analysis of the SMB process was performed numerically using a well-established model.     

Rotational fluctuations of water inside the nanopores of SBA-type molecular sieves

The rotational molecular dynamics of water confined to nanoporous molecular sieves of a regular hexagonal (SBA-15) and of a foamlike pore structure was studied by dielectric spectroscopy in the frequency range from 10(-2) to 10(9) Hz and in a broad temperature interval. Two relaxation processes were observed: the process at lower frequencies is related to water molecules forming a layer, which is strongly adsorbed at the pore surface, whereas the relaxation process at higher frequencies is assigned to fluctuations of water molecules situated close to the center of the pore. The relaxation times of the low-frequency process for both materials and of the high-frequency process for the SBA-15 material have an unusual saddlelike temperature dependence, reported here for the first time. To describe this temperature dependence, a model developed for water confined to nanoporous glasses by Ryabov et al. [J. Phys. Chem. B 2001, 105, 1845] was applied, which considers two competing effects. The characteristic features of these two competing processes were compared with those reported for other porous systems.     

Detection of peonidin and pelargonidin glycosides in black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) by high-performance liquid chromatography/electrospray ionization mass spectrometry

Anthocyanins of black carrots (Daucus carota ssp. sativus var. atrorubens Alef.) were investigated by high-performance liquid chromatography/electrospray ionization mass spectrometry. Besides the main compounds which have already been characterized in detail as cyanidin glycosides, six anthocyanins with m/z 757, 565, 595, 903, 933 and 903 were detected and unambiguously characterized as peonidin and pelargonidin glycosides, based on their fragmentation patterns. Peonidin and pelargonidin were identified after acid hydrolysis by comparison of the fragmentation patterns and retention times of the released aglycones with those of standard compounds. To the best of our knowledge this is the first report on anthocyanins in black carrots consisting of aglycones other than cyanidin.     

Syntheses, structures, and surface aromaticity of the new carbaalane [(AlH)(6)(AlNMe(3))(2)(CCH(2)R)(6)] (R = Ph, CH(2)SiMe(3)) and a stepwise functionalization of the inner and outer sphere of the cluster

The reaction of the acetylene RC triple bond CH (R = Ph, CH(2)SiMe(3)) with an excess of AlH(3).NMe(3) in boiling toluene leads to the carbaalane [(AlH)(6)(AlNMe(3))(2)(CCH(2)R)(6)] (R = Ph 1, CH(2)SiMe(3) 2) in good yield. Treatment of 2 with BCl(3) under varying conditions gives the chlorinated products [(AlCl)(6)(AlNMe(3))(2)(CCH(2)CH(2)SiMe(3))(6)] 3 and [(AlCl)(6)(AlNMe(3))(2)(CCH(2)CH(2)SiMe(2)Cl)(6)] 4, respectively. The latter clearly demonstrates that the cluster can be stepwise functionalized within the inner and outer sphere. The X-ray single-crystal structures of 1, 2, and 4 have been determined. All compounds have in common that the central core consists of a cluster having eight aluminum and six carbon atoms. The bonding properties in this cluster are described as a new manifestation of three-dimensional surface aromaticity. Each Al(4)C fragment of the cube is formed by four bonds with three electron pairs, thus leading to a strong delocalization of the electrons. A phenomenological modeling using a three-dimensional Hückel scheme with fitted parameters to reproduce the energies from ab initio calculations revealed that the orbital scheme localized at one Al(4)C fragment possesses an orbital sextet with a large HOMO-LUMO gap. This is in line with the criteria of aromaticity. The idea of aromaticity was sustained also by qualitative valence bond reasons enumerating the different resonance structures by means of graph theoretical methods.     

Coupled liquid chromatography-gas chromatography for the rapid analysis of gamma-oryzanol in rice lipids

An approach based on on-line coupled liquid chromatography-gas chromatography (LC-GC) was developed for the rapid analysis of gamma-oryzanol in rice. Total lipids were extracted from rice and subjected to LC-GC without any prior purification. gamma-Oryzanol was pre-separated by HPLC from rice lipids and transferred on-line to GC analysis in order to separate its major constituents. 24-methylenecycloartanyl ferulate, cycloartenyl ferulate, campesteryl ferulate, beta-sitosteryl ferulate and campestanyl ferulate. The identities of the compounds were confirmed by off-line GC-MS analysis. Total gamma-oryzanol content could be quantified by HPLC-UV detection and the distribution of gamma-oryzanol constituents could be determined by on-line coupled GC analysis. The proposed methodology paves the way for high-throughput investigations providing information on natural variations in gamma-oryzanol content and its composition in different rice varieties.     

Synthesis and immobilization of quaternary ammonium cations in acidic zeolites

A general method for the synthesis of quaternary ammonium cations in acidic zeolites by a direct reaction of tertiary amines and alcohols is described.