End-labeled free-solution electrophoresis of DNA

DNA is a free-draining polymer. This subtle but "unfortunate" property of highly charged polyelectrolytes makes it impossible to separate nucleic acids by free-flow electrophoresis. This is why one must typically use a sieving matrix, such as a gel or an entangled polymer solution, in order to obtain some electrophoretic size separation. An alternative approach consists of breaking the charge to friction balance of free-draining DNA molecules. This can be achieved by labeling the DNA with a large, uncharged molecule (essentially a hydrodynamic parachute, which we also call a drag-tag) prior to electrophoresis; the resulting methodology is called end-labeled free-solution electrophoresis (ELFSE). In this article, we review the development of ELFSE over the last decade. In particular, we examine the theoretical concepts used to predict the ultimate performance of ELFSE for single-stranded (ssDNA) sequencing, the experimental results showing that ELFSE can indeed overcome the free-draining issue raised above, and the technological advances that are needed to speed the development of competitive ELFSE-based sequencing and separation technologies. Finally, we also review the reverse process, called free-solution conjugate electrophoresis (FSCE), wherein uncharged polymers of different sizes can be analyzed using a short DNA molecule as an electrophoretic engine.     

Synthesis, characterization and Schlenk equilibrium studies of methylmagnesium compounds with O- and N-donor ligands - the unexpected behavior of [MgMeBr(pmdta)] (pmdta = N,N,N′,N″,N″-pentamethyldiethylenetriamine)

MgMe₂ (1) was found to react with 1,4-diazabicyclo[2.2.2]octane (dabco) in tetrahydrofuran (thf) yielding a binuclear complex [{MgMe₂(thf)}₂(μ-dabco)] (2). Furthermore, from reactions of MgMeBr with diglyme (diethylene glycol dimethyl ether), NEt₃, and tmeda (N,N,N′,N′-tetramethylethylenediamine) in etheral solvents compounds MgMeBr(L), (L = diglyme (5); NEt₃ (6); tmeda (7)) were obtained as highly air- and moisture-sensitive white powders. From a thf solution of 7 crystals of [MgMeBr(thf)(tmeda)] (8) were obtained. Reactions of MgMeBr with pmdta (N,N,N′,N″,N″-pentamethyldiethylenetriamine) in thf resulted in formation of [MgMeBr(pmdta)] (9) in nearly quantitative yield. On the other hand, the same reaction in diethyl ether gave MgMeBr(pmdta) · MgBr₂(pmdta) (10) and [{MgMe₂(pmdta)}₇{MgMeBr(pmdta)}] (11) in 24% and 2% yield, respectively, as well as [MgMe₂(pmdta)] (12) as colorless needle-like crystals in about 26% yield. The synthesized methylmagnesium compounds were characterized by microanalysis and ¹H and ¹³C NMR spectroscopy. The coordination-induced shifts of the ¹H and ¹³C nuclei of the ligands are small; the largest ones were found in the tmeda and pmdta complexes. Single-crystal X-ray diffraction analyses revealed in 2 a tetrahedral environment of the Mg atoms with a bridging dabco ligand and in 8 a trigonal-bipyramidal coordination of the Mg atom. The single-crystal X-ray diffraction analyses of [MgMe₂(pmdta)] (12) and [MgBr₂(pmdta)] (13) showed them to be monomeric with five-coordinate Mg atoms. The square-pyramidal coordination polyhedra are built up of three N and two C atoms in 12 and three N and two Br atoms in 13. The apical positions are occupied by methyl and bromo ligands, respectively. Temperature-dependent ¹H NMR spectroscopic measurements (from 27 to −80 °C) of methylmagnesium bromide complexes MgMeBr(L) (L = thf (4); diglyme (5); NEt₃ (6); tmeda (7)) in thf-d₈ solutions indicated that the deeper the temperature the more the Schlenk equilibria are shifted to the dimethylmagnesium/dibromomagnesium species. Furthermore, at −80 °C the dimethylmagnesium compounds are predominant in the solutions of Grignard compounds 4-6 whereas in the case of the tmeda complex7 the equilibrium constant was roughly estimated to be 0.25. In contrast, [MgMeBr(pmdta)] (9) in thf-d₈ revealed no dismutation into [MgMe₂(pmdta)] (12) and [MgBr₂(pmdta)] (13) even up to −100 °C. In accordance with this unexpected behavior, 1:1 mixtures of 12 and 13 were found to react in thf at room temperature yielding quantitatively the corresponding Grignard compound 9. Moreover, the structures of [MgMeBr(pmdta)] (9c), [MgMe₂(pmdta)] (12c), and [MgBr₂(pmdta)] (13c) were calculated on the DFT level of theory. The calculated structures 12c and 13c are in a good agreement with the experimentally observed structures 12 and 13. The equilibrium constant of the Schlenk equilibrium (2 9c ? 12c + 13c) was calculated to be K_gas = 2.0 × 10⁻³ (298 K) in the gas phase. Considering the solvent effects of both thf and diethyl ether using a polarized continuum model (PCM) the corresponding equilibrium constants were calculated to be K_thf = 1.2 × 10⁻³ and K_ether = 3.2 × 10⁻³ (298 K), respectively.     

Photodynamic treatment with fractionated light decreases production of reactive oxygen species and cytotoxicity in vitro via regeneration of glutathione

Photodynamic therapy removes unwanted or harmful cells by overproduction of reactive oxygen species (ROS). Fractionated light delivery in photodynamic therapy may enhance the photodynamic effect in tumor areas with insufficient blood supply by enabling the reoxygenation of the treated area. This study addresses the outcome of fractionated irradiation in an in vitro photodynamic treatment (PDT) system, where deoxygenation can be neglected. Our results show that fractionated irradiation with light/dark intervals of 45/60 s decreases ROS production and cytotoxicity of PDT. This effect can be reversed by addition of 1,3-bis-(2-chlorethyl)-1-nitrosurea (BCNU), an inhibitor of the glutathione reductase. We suggest that the dark intervals during irradiation allow the glutathione reductase to regenerate reduced glutathione (GSH), thereby rendering cells less susceptible to ROS produced by PDT compared with continuous irradiation. Our results could be of particular clinical importance for photodynamic therapy applied to well-oxygenated tumors.     

Commercially available 5'-DMT phosphoramidites as reagents for the synthesis of vinylphosphonate-linked oligonucleic acids

[reaction: see text]. Commercially available cyanoethyl phosphoramidites derived from T, d(C), d(A), and d(G) were hydrolyzed (1H-tetrazole, MeCN/H2O) to give the corresponding H-phosphonates in excellent yields. Palladium(0)-catalyzed cross-coupling of each of these with the thymidine-derived vinylbromide 2 afforded the corresponding vinylphosphonate-linked dimers TT, d(C)T, d(A)T, and d(G)T in modest to good yields. The TT dimer was further elaborated to give a 5'-DMT-TT-3'-CEP building block, and this was used in the automated synthesis of the TpTTpT tetramer.     

Organic synthesis on the microgram scale. III separation and purification of steroids

Summary This paper describes techniques for separation and purification of steroids on the thin-layer plate. Two or more operations can be performed on the same plate in order to avoid loss of material and product.When a steroid diketone (progesterone) is treated with 2,4-dinitrophenylhydrazine on silica gel, three products are obtainable. These compounds can be readily purified on the same thin-layer plate and converted to the parent steroid by means of stannous chloride in dilute hydrochloric acid.

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Zusammenfassung Trennung und Reinigung von Steroiden auf der Dünnschichtplatte werden beschrieben. Zwei oder mehr Operationen lassen sich, um Substanzverluste zu vermeiden, auf derselben Platte ausführen.Wird ein Diketosteroid (Progesteron) mit 2,4-Dinitrophenylhydrazin auf Kieselgel behandelt, so erhält man drei Reaktionsprodukte, die auf derselben Platte leicht gereinigt und mit Zinn(II)-chlorid in verd. Salzsäure in das entsprechende Ausgangssteroid zurückverwandelt werden können.

     

Novel rhenium chelate system derived from dimercaptosuccinic acid for the selective labeling of biomolecules

This work is part of an effort to develop chelating agents for stable binding and easy conjugation of Re-188 to biologically interesting structures. Starting from the well-known in vivo stability of [(188)ReO(DMSA)(2)](-), we want to exploit this coordination system for the design of (188)ReO(V) chelates, which are stable toward reoxidation to perrhenate and toward ligand exchange under all conditions of radiopharmaceutical development. Therefore, a new type of tetradentate ligand has been synthesized by bridging two molecules of N,N'-diisobutyl-2,3-dimercaptosuccinamide with N-(3-aminopropyl)propane-1,3-diamine. The resulting stereoisomeric tetrathiolato S(4) ligand of composition ((i)()Bu)(2)N(O)C-C(SH)-C(SH)-C(O)NH-(CH(2))(3)-NH-(CH(2))(3)-NHC(O)-C(SH)-C(SH)-C(O)N((i)Bu)(2) forms anionic five-coordinate oxorhenium(V) complexes by a ligand-exchange reaction of NBu(4)[ReOCl(4)] in methanol. In the absence of a base, the compounds were isolated as "betaine", [ReO(S(4))], with the protonated nitrogen of the bridge serving as an internal "counterion". Two representatives have been fully characterized in both the solid and solution states and found to adopt the expected square-pyramidal coordination geometry. The equatorial plane is formed by four thiolate sulfur atoms, whereas the oxygen occupies the apical position. The orientation of the metal oxo group is exo in relation to the carbamido groups in both isomers. Both complexes are stereoisomeric regarding the junction of the triamine chain.     

Bead cellulose derivatives as supports for immobilization and chromatographic purification of proteins

Characteristic data are presented for Divicell, a macroporous bead cellulose with excellent flow parameters. The preparation of Divicell derivatives and their properties are described with respect to their application as chromatographic supports. The ion exchangers Divicell DEAE and Divicell CM were manufactured in two types with different exclusion limits and an available capacity for proteins of up to 100 mg/ml gel. Divicell Blue is a bead cellulose with covalently bound Cibacron Blue F3G-A and was found to be a very suitable adsorbent for the selective separation and purification of human serum albumin. Activation of Divicell with sodium periodate, epichlorohydrin and 5-norbornene-2,3-dicarboximido carbonochloridate provided activated supports used for immobilization of ligands in organic solvents and in aqueous solutions. Coupling of amines, diamines, amino acids, carbohydrates and proteins is described. The immobilized ligands retained their biological activity as determined by their specific adsorption of proteins. Divicell alkyl derivatives were tested in hydrophobic interaction chromatography with bovine serum albumin as a model. Examples are presented of the application of Divicell derivatives to the purification of biomacromolecules such as immunoglobulins and lectins by affinity chromatography. The results were comparable to those obtained using the corresponding Sepharose-derived absorbents.     

Nucleation and growth of germanium nanowires seeded by organic monolayer-coated gold nanocrystals

Germanium nanowires, ranging from 10 to 150 nm in diameter, were grown several micrometers in length in cyclohexane heated and pressurized above its critical point. Alkanethiol-protected gold nanocrystals, either 2.5 or 6.5 nm in diameter, were used to seed wire formation. Growth proceeded through a solution-liquid-solid mechanism at growth temperatures ranging from 300 to 450 degrees C. At temperatures exceeding 500 degrees C, large Ge particulates formed due to unfavorable growth kinetics. Temperature, the nature of the precursor, precursor concentration, and the Au:Ge ratio were determining factors in nanowire morphology. The Ge nanowires were characterized using a range of techniques, including XPS, XRD, high-resolution TEM and SEM, nanometer-scale EDS mapping, and DTA.