A rapid and sensitive CE method with field-enhanced sample injection and in-capillary derivatization for selenomethionine metabolism catalyzed by flavin-containing monooxygenases

A rapid and sensitive electrophoretically mediated microanalysis method with field-enhanced sample injection (FESI) for in-capillary derivatization was developed to determine selenomethionine (SeMet) and selenomethionine selenoxide (SeOMet). Phthalic anhydride (PA) was selected as the derivatization reagent due to the fast reaction at room temperature and the stability of derivatives. The in-capillary derivatization was accomplished by electrophoretically mixing PA and sample plugs. PA reagent was introduced hydrodynamically into the capillary, whereas the sample solution was injected electrokinetically, thus allowing a selective preconcentration of the analytes by FESI. For FESI, the optimum sample solvent was 2 mM borate solution. The borate buffer was suitable for both in-capillary derivatization and separation of the derivatives. The combination of electrophoretically mediated microanalysis with FESI for in-capillary derivatization was successfully achieved with about 800-fold concentration sensitivity enhancement compared to direct CE-UV detection in the same setup. The present method is miniaturized and fully automated, which ensures the on-line derivatization, stacking, separation and detection in 10 min. Finally, the developed method was successfully applied to measure enzyme activities by analyzing the reaction mixtures of SeMet with human flavin-containing monooxygenases (FMO). The results showed that both FMO1 and FMO3, but not FMO5 could catalyze the Se-oxygenation of SeMet.     

A novel (18)O inverse labeling-based workflow for accurate bottom-up mass spectrometry quantification of proteins separated by gel electrophoresis

In the present work we report on a novel and fast protocol for accurate bottom-up protein quantification that overcomes the drawbacks of in-gel digestion and MALDI analysis, while maintaining their benefits. It relies on the following steps: (i) gel electrophoresis separation of proteins, (ii) fast in-gel protein digestion with trypsin, (iii) (18)O-labeling through the decoupled method, (iv) quantification through selected peptides previously chosen using the (18)O inverse labeling approach and that, finally, (v) it takes advantage of software specifically developed to select the peptides that will drive the quantification of the protein in an automated mode. We have accurately quantified the following six proteins: glycogen phosphorylase, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor, and α-lactalbumin. As a case study we have quantified the protein vitellogenin in plasma of Cyprinus carpio exposed to high levels of estrogens. The proposed new protocol was validated against the traditional ELISA method; both were found to provide comparable results (non-parametric Mann-Whitney U-test).     

Global ab initio potential energy surfaces for the O2(3Σg-)+O2(3Σg-) interaction

Completely ab initio global potential energy surfaces (PESs) for the singlet and triplet spin multiplicities of rigid O(2)((3)Σ(g)(-))+O(2)((3)Σ(g)(-)) are reported for the first time. They have been obtained by combining an accurate restricted coupled cluster theory with singles, doubles, and perturbative triple excitations [RCCSD(T)] quintet potential [Bartolomei et al., J. Chem. Phys. 128, 214304 (2008)] with complete active space second order perturbation theory (CASPT2) or, alternatively, multireference configuration interaction (MRCI) calculations of the singlet-quintet and triplet-quintet splittings. Spherical harmonic expansions, containing a large number of terms due to the high anisotropy of the interaction, have been built from the ab initio data. The radial coefficients of these expansions are matched at long range distances with analytical functions based on recent ab initio calculations of the electric properties of the monomers [M. Bartolomei, E. Carmona-Novillo, M. I. Hernández, J. Campos-Martínez, and R. Hernández-Lamoneda, J. Comput. Chem. (2010) (in press)]. The singlet and triplet PESs obtained from either RCCSD(T)-CASPT2 or RCCSD(T)-MRCI calculations are quite similar, although quantitative differences appear in specific terms of the expansion. CASPT2 calculations are the ones giving rise to larger splittings and more attractive interactions, particularly in the region of the absolute minima (in the rectangular D(2h) geometry). The new singlet, triplet, and quintet PESs are tested against second virial coefficient B(T) data and, their spherically averaged components, against integral cross sections measured with rotationally hot effusive beams. Both types of multiconfigurational approaches provide quite similar results, which, in turn, are in good agreement with the measurements. It is found that discrepancies with the experiments could be removed if the PESs were slightly more attractive. In this regard, the most attractive RCCSD(T)-CASPT2 PESs perform slightly better than the RCCSD(T)-MRCI counterpart.     

Evaluation of dispersive liquid-liquid microextraction for the simultaneous determination of chlorophenols and haloanisoles in wines and cork stoppers using gas chromatography-mass spectrometry

Dispersive liquid-liquid microextraction (DLLME) coupled with gas chromatography-mass spectrometry (GC-MS) was evaluated for the simultaneous determination of five chlorophenols and seven haloanisoles in wines and cork stoppers. Parameters, such as the nature and volume of the extracting and disperser solvents, extraction time, salt addition, centrifugation time and sample volume or mass, affecting the DLLME were carefully optimized to extract and preconcentrate chlorophenols, in the form of their acetylated derivatives, and haloanisoles. In this extraction method, 1mL of acetone (disperser solvent) containing 30μL of carbon tetrachloride (extraction solvent) was rapidly injected by a syringe into 5mL of sample solution containing 200μL of acetic anhydride (derivatizing reagent) and 0.5mL of phosphate buffer solution, thereby forming a cloudy solution. After extraction, phase separation was performed by centrifugation, and a volume of 4μL of the sedimented phase was analyzed by GC-MS. The wine samples were directly used for the DLLME extraction (red wines required a 1:1 dilution with water). For cork samples, the target analytes were first extracted with pentane, the solvent was evaporated and the residue reconstituted with acetone before DLLME. The use of an internal standard (2,4-dibromoanisole) notably improved the repeatability of the procedure. Under the optimized conditions, detection limits ranged from 0.004 to 0.108ngmL(-1) in wine samples (24-220pgg(-1) in corks), depending on the compound and the sample analyzed. The enrichment factors for haloanisoles were in the 380-700-fold range.     

Non-nucleoside building blocks for copper-assisted and copper-free click chemistry for the efficient synthesis of RNA conjugates

Novel non-nucleoside alkyne monomers compatible with oligonucleotide synthesis were designed, synthesized, and efficiently incorporated into RNA and RNA analogues during solid-phase synthesis. These modifications allowed site-specific conjugation of ligands to the RNA oligonucleotides through copper-assisted (CuAAC) and copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reactions. The SPAAC click reactions of cyclooctyne-oligonucleotides with various classes of azido-functionalized ligands in solution phase and on solid phase were efficient and quantitative and occurred under mild reaction conditions. The SPAAC reaction provides a method for the synthesis of oligonucleotide-ligand conjugates uncontaminated with copper ions.     

Tautomerism and microsolvation in 2-hydroxypyridine/2-pyridone

The Fourier transform microwave spectra of the hydrated forms of the tautomeric pair 2-pyridinone/2-hydroxypyridine (2PO/2HP) have been investigated in a supersonic expansion. Three hydrated species, 2PO-H?O, 2HP-H?O, and 2PO-(H?O)?, have been observed in the rotational spectrum. Each molecular complex was confidently identified by the features of the 1?N quadrupole hyperfine structure of the rotational transitions. The presence of water affects the tautomeric equilibrium -N═C(OH)- ? -NH-C(═O)-, which is shifted to the enol form for the bare molecules 2PO/2HP but to the keto tautomer for the hydrated forms.     

A neutral zwitterionic molecular solid

We report on the acid ethylenedithiotetrathiafulvaleneamidoglycine (EDT-TTF-CO-NH-CH(2)-CO(2)H; 1; EDT-TTF=ethylenedithiotetrathiafulvalene) and the 1:1 adduct [(EDT-TTF)(·+)-CO-NH-CH(2)-(CO(2))(-)][(EDT-TTF)-CO-NH-CH(2)-(CO(2)H)]·CH(3)OH (2), a new type of hydrogen-bonded, 1:1 acid/zwitterion hybrid embrace of redox peptidics into a two-dimensional architecture, an example of a system deliberately fashioned so that oxidation of π-conjugated cores toward the radical-cation form would interfere with the activity of the appended ionizable residues in the presence of a templating base during crystal growth. First-principles calculations demonstrate that, notwithstanding preconceived ideas, a metallic state is more stable than the hole-localized alternatives for a neat 1:1 neutral acid/zwitterion hybrid. The inhomogeneous Coulomb field associated with proton-shared, interstacks O-H···O hydrogen bonds between the ionizable residues distributed on both sides of the two-dimensional π-conjugated framework leads, however, to a weak hole localization responsible for the activated but high conductivity of 1 S cm(-1). This situation is reminiscent of the role of the environment on electron transfer in tetraheme cytochrome c, in which the protonation state of a heme propionate becomes paramount, or ion-gated transport phenomena in biology. These observations open rather intriguing opportunities for the construction of electronic systems at the interface of chemistry and biology.     

Phosphine and Thiophene Cyclopalladated Complexes: Hydrolysis Reactions in Strong Acidic Media

The mechanisms for the hydrolysis of organopalladium complexes [Pd(CNN)R]BF₄ (R=P(OPh)₃, PPh₃, and SC₄H₈) were investigated at 25 °C by using UV/Vis absorbance measurements in 10 % v/v ethanol/water mixtures containing different sulphuric acid concentrations in the 1.3–11.7 M range. In all cases, a biphasic behavior was observed with rate constants k_1obs, which corresponds to the initial step of the hydrolysis reaction, and k_2obs, where k_1obs>k_2obs. The plots of k_1obs and k_2obs versus sulfuric acid concentration suggest a change in the reaction mechanism. The change with respect to the k_1obs value corresponds to 35 %, 2 %, and 99 % of the protonated complexes for R=PPh₃, P(OPh)₃, and SC₄H₈, respectively. Regarding k_2obs, the change occurred in all cases at about 6.5 M H₂SO₄ and matched up with the results reported for the hydrolysis of the 2‐acetylpyridinephenylhydrazone (CNN) ligand. By using the excess acidity method, the mechanisms were elucidated by carefully looking at the variation of k_i,_obs (i=1,2) versus ${c_{{\rm{H}}^ + } }$ . The rate‐determining constants, k_0,A‐1, k_0,A‐2, and k_0,A‐SE2 were evaluated in all cases. The R=P(OPh)₃ complex was most reactive due to its π‐acid character, which favors the rupture of the trans nitrogen–palladium bond in the A‐2 mechanism and also that of the pyridine nitrogen–palladium bond in the A‐1 mechanism. The organometallic bond exerts no effect on the relative basicity of the complexes, which are strongly reliant on the substituent.     

Complexation of Hg2+ with α‐Lipoic and Dihydrolipoic Acids: Study by Differential Pulse Voltammetry on Rotating Au‐Disk Electrode and ESI‐MS

The complexation of the natural antioxidants α‐lipoic acid (ALA) and its reduced form dihydrolipoic acid (DHLA) with Hg²⁺ was investigated by a recently proposed differential pulse voltammetric (DPV) method using the rotating Au‐disk electrode. Complexation processes are proposed from the multivariate curve resolution by alternating least squares (MCR‐ALS) analysis of DPV titration data. Main complexes were both 1 : 1 Hg : ALA and Hg : DHLA, although the formation of 1 : 2 complexes can be also deduced. ALA and DHLA show different Hg²⁺‐binding patterns at different pH. Voltammetric findings are completed with the data obtained by electrospray ionization mass‐spectrometry (ESI‐MS), especially in negative mode.