Template synthesis in the nickel(II)–thiocarbohydrazide–propanone triple system

The complexing process proceeding in the Ni^II–thiocarbohydrazide (H₂N–H–NC(=S)–NH–NH₂)–propanone triple system in EtOH solution and nickel(II)hexacyanoferrate(II) gelatin-immobilized matrix has been studied. It has been found that in the first case, template synthesis leading, as a minimum, to formation of three coordination compounds of Ni^II with (N,N,S,S)-donor tetradentate ligands having NiL¹, NiL² and NiL³compositions where L¹ is 4,6,6-trimethyl-2,3,7,8-tetraazanonen-3-di(thiohydrazide)-1,9, L² is 4,6,6,12-tetrametyl-1,9-dithio-2,3,7,8,10,11-hexaazatridekadien-3,11-hydrazide-1 and L³ is 2,8,10,10,16-pentamethyl-5,13-dithio-3,4,6,7,11,12,14,15-octaazaheptadekatrien-2,7,15 is observed, whereas in the gelatin-immobilized matrix, a complexing process in the system considered does not occur.     

Effect of Cysteic Acid Position on the Negative Ion Fragmentation of Proteolytic Derived Peptides

A study on the effect of cysteic acid position on the types of fragment ions formed by collision-induced dissociation (CID) of [M – H]⁻ ions is presented. Of particular note is the observation of d-type fragment ions for peptides that contain an N-terminal cysteic acid (fixed negative charge) and cleavable amino acid side chains possessing a β-γ carbon–carbon bond. For example, the CID mass spectrum of oxidized cys-kemptide (C_oxLRRASLG) [M – H + O₃]⁻ ions contains abundant series of d-type fragment ions, and similar results are observed for oxidized cysteine-containing ribonuclease A proteolytic peptides. The d _i fragment ions are assumed to arise by a charge-remote and/or charge-assisted fragmentation mechanism, which both occur at high collision energies and involve consecutive reactions (i.e., the formation of a _i ions followed by the elimination of the side chain to form d _i ions).     

Towards liquid chromatography time-scale peptide sequencing and characterization of post-translational modifications in the negative-ion mode using electron detachment dissociation tandem mass spectrometry

Electron detachment dissociation (EDD) of peptide poly-anions is gentle towards post-translational modifications (PTMs) and produces predictable and interpretable fragment ion types (a., x ions). However, EDD is considered an inefficient fragmentation technique and has not yet been implemented in large-scale peptide characterization strategies. We successfully increased the EDD fragmentation efficiency (up to 9%), and demonstrate for the first time the utility of EDD-MS/MS in liquid chromatography time-scale experiments. Peptides and phosphopeptides were analyzed in both positive- and negative-ion mode using electron capture/transfer dissociation (ECD/ETD) and EDD in comparison. Using approximately 1 pmol of a BSA tryptic digest, LC-EDD-MS/MS sequenced 14 peptides (27% aa sequence coverage) and LC-ECD-MS/MS sequenced 19 peptides (39% aa sequence coverage). Seven peptides (18% aa sequence coverage) were sequenced by both EDD and ECD. The relative small overlap of identified BSA peptides demonstrates the complementarity of the two dissociation modes. Phosphopeptide mixtures from three trypsin-digested phosphoproteins were subjected to LC-EDD-MS/MS resulting in the identification of five phospho-peptides. Of those, one was not found in a previous study using a similar sample and LC-ETD-MS/MS in the positive-ion mode. In this study, the ECD fragmentation efficiency (15.7% av.) was superior to the EDD fragmentation efficiency (3.6% av.). However, given the increase in amino acid sequence coverage and extended PTM characterization the new regime of EDD in combination with other ion-electron fragmentation techniques in the positive-ion mode is a step towards a more comprehensive strategy of analysis in proteome research.     

Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen

A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)₂ and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[α]-carbazol-11-yl) ethyl chloroformate (BCEC–Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[α]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC–Cl. The new reagent BCEC–Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC–amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)₂ and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I _BCEC/I _BCEOC = 1.17–3.57, I _BCEC/I _FMOC = 1.13–8.21, and UV_BCEC/UV_BCEOC = 1.67–4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C₁₈ column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)₂. Excellent linear responses were observed, with coefficients of >0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)₂ and Try from bee-collected pollen (bee pollen) samples.     

Trennung,Identifizierung und Bestimmung von Bactericiden auf der Basis von halogenierten Aromaten mittels Gas-Chromatographie

The separation of a mixture of 22 bactericides has been achieved by gas chromatography on columns with silicone rubber W-982 as stationary phase with temperatures between 100° and 300°C. The unchanged compounds as well as their silylation products have been used. The latter are more conveniently used especially for the quantitative determination. To be able to calculate the retention indices after Kovats gas chromatography has been performed isothermally at 180°C for the more volatile compounds and at 250°C for all other bactericides.The retention indices obtained under these conditions are tabulated together with the limits of detection.     

A study of noncovalent complexes involving single-stranded DNA and polybasic compounds using nanospray mass spectrometry

Noncovalent complexes involving a single-stranded DNA oligonucleotide and a polybasic compound (spermine, penta-L-lysine, penta-L-arginine, or polydisperse poly-L-lysine) were detected by nanospray-MS. Several control experiments tended to show that these complexes preexisted in solution and that the interactions were initially ionic ones between oligonucleotide phosphates and protonated basic sites of the polybasic compound. Collision-induced dissociation (CID) experiments carried out with these complexes allowed us to identify some differences in the nature of the interactions between the solution and the gas phase, arising from possible proton transfers. Different dissociation pathways were observed according to the nature of the polybasic compound and to the initial charge state of the complex. The complex involving spermine dissociated by cleavage of noncovalent bonds leading to the separation of the two components, whereas the one involving penta-L-arginine underwent fragmentations of covalent bonds. Both behaviors were independent of the initial charge state of the complex. On the other hand, the dissociation pathway of the complex involving penta-L-lysine has been shown to be clearly charge state dependent. Noncovalent dissociation (separation of the two components) driven by coulomb repulsion occurred for the higher charged complexes, whereas fragmentation of covalent bonds was the main pathway of the lower charged complexes. In the latter case, differences in CID behavior were observed for different lengths of poly-L-lysine.     

A label-free sensing method for phosphopeptides using two-layer gold nanoparticle-based localized surface plasma resonance spectroscopy

In this study, a new type of localized surface plasmon resonance (LSPR) sensing substrate for phosphopeptides was explored. It has been known that LSPR response for target species is larger in the near-infrared region (NIR) than in the visible region of the electromagnetic spectrum. Several types of noble metal nanoparticles (NPs) with NIR absorption capacities have been previously demonstrated as effective LSPR-sensing nanoprobes. Herein, we demonstrate a straightforward approach with improved sensitivity by simply using layer-by-layer (LBL) spherical Au NPs self-assembled on glass slides as the LSPR-sensing substrates that are responsive in the NIR region of the electromagnetic spectrum. The modified glass slide acquired an LSPR absorption band in the NIR, which resulted from the dipole–dipole interactions between Au NPs. To enable the chip to sense phosphopeptides, the surface of the glass chip was spin-coated with thin titania film (TiO₂-Glass@Au NPs). Absorption spectrophotometry was employed as a detection tool. Tryptic digest of α-casein was used as a model sample. The feasibility of using the new LSPR approach for detecting a potential risk factor leading to cancers (i.e., phosphorylated fibrinopeptide A) directly from human serum samples was demonstrated. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was used to confirm the results.     

Immobilization of <Emphasis Type="Italic">Escherichia coli</Emphasis> novablue γ-glutamyltranspeptidase in Ca-alginate-<Emphasis Type="Italic">k</Emphasis>-carrageenan beads

The recombinant Escherichia coli gamma-glutamyltranspeptidase (EcGGT) was immobilized in Ca-alginate-kappa-carrageenan beads. Effects of alginate concentration, amount of loading enzyme, and bead size on the entrapped activity were investigated. Optimum alginate concentration for EcGGT immobilization was found to be 2% (w/v). Using a loading enzyme concentration of 1.5 mg/g alginate, maximum enzyme activity was observed. With increase in bead size from 1.9 to 3.1 mm, the immobilization efficiency was decreased significantly because of mass transfer resistance. Thermal stability of the free EcGGT was increased as a result of the immobilization. Ca-alginate-kappa-carrageenan-EcGGT beads were suitable for up to six repeated uses, losing only 45% of their initial activity. Upon 30 days of storage the preserved activity of free and immobilized enzyme were found as 4% and 68%, respectively. The synthesis of L: -theanine was performed in 50 mM Tris-HCl buffer (pH 10) containing 25 mM L: -glutamine, 40 mM ethylamine, and 1.5 mg EcGGT/g alginate at 40 degrees C for 12 h, and a conversion rate of 27% was achieved.     

Unusual reaction of chloroacetyl chloride with 1,2-dichloroethene. Synthesis and properties of 2-chlorovinyl dichloromethyl ketone

The reaction of chloroacetyl chloride with 1,2-dichloroethene in the presence of AlCl₃ unexpectedly led to the formation of (E)-1,1,4-trichlorobut-3-en-2-one whose structure was proved by ¹H and ¹³C NMR, IR, and mass spectra and independent synthesis. A probable reaction scheme was proposed, which involves transformation of initially formed 1,2,4-trichloro-3-oxobutan-2-yl cation by the action of AlCl₃. The high reactivity of the vinylic halogen atom in (E)-1,1,4-trichlorobut-3-en-2-one was demonstrated by its reactions with nitrogen-centered nucleophiles (triethylamine, aniline, 3,5-dimethyl-1H-pyrazole) and sodium sulfide. These reactions involved only the C-Cl bond in the vinyl fragment and afforded (4,4-dichloro-3-oxobut-2-en-1-yl)triethylammonium chloride, 1,1-dichloro-4-phenylaminobut-3-en-2-one, 1-(4,4-dichloro-3-oxobut-2-en-1-yl)-3,5-dimethyl-1H-pyrazole, and 4,4′-thiobis(1,1-dichlorobut-3-en-2-one), respectively. The reaction of 1,1,4-trichlorobut-3-en-2-one with benzylhydrazine gave a mixture of 1,3- and 1,5-disubstituted pyrazoles.