Characterization of alkyl-cobalamins formed on trapping of epoxide metabolites of 1,3-butadiene

Analytical methods facilitating studies of electrophilically reactive and genotoxic compounds in vitro and in vivo are needed. The strong nucleophile, cob(I)alamin, formed by reduction of Vitamin B12 [cob(III)alamin], may be used for trapping and analysis of 1,2-epoxides and other electrophiles. In the present study, cob(I)alamin is evaluated as an analytical tool for 1,2-epoxide metabolites (oxiranes) of 1,3-butadiene. Products of reaction of cob(I)alamin with 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EBdiol) have been analyzed by reversed phase high performance liquid chromatography (HPLC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) and ultraviolet diode array detection (UV-DAD). It was shown that a specific alkyl-CbI complex is formed for each metabolite and that it was possible to discriminate between the products by HPLC-UV and by LC-MS. Quantification of DEB with the method by use of another 1,2-epoxide as an internal standard was successfully performed. The possibility of using cob(I)alamin for trapping and analysis of the three oxirane metabolites of 1,3-butadiene will facilitate quantitative comparisons of species in vitro with regard to metabolism of 1,3-butadiene.     

Triplet-doublet electronic energy transfer from benzophenone to the ketyl radical: viscosity and magnetic field effects

The influence of solvent viscosity and an external magnetic field on the rate constant of electronic energy transfer from triplet bonzophenone to the ketyl radical is studied; it is concluded that the transfer is mediated by electron exchange.     

High-efficiency energy transfer from carotenoids to a phthalocyanine in an artificial photosynthetic antenna

Two carotenoid pigments have been linked as axial ligands to the central silicon atom of a phthalocyanine derivative, forming molecular triad 1. Laser flash studies on the femtosecond and picosecond time scales show that both the carotenoid S1 and S2 excited states act as donor states in 1, resulting in highly efficient singlet energy transfer from the carotenoids to the phthalocyanine. Triplet energy transfer in the opposite direction was also observed. In polar solvents efficient electron transfer from a carotenoid to the phthalocyanine excited singlet state yields a charge-separated state that recombines to the ground state of 1.     

Bridgehead nitrogen heterocycles which contain the quinazoline moiety -- synthesis and cycloaddition of 1,2-dihydroquinazoline 3-oxides

A novel synthesis of 1,2-disubstituted 1,2-dihydroquinazoline 3-oxides 8 and the first ever examples of 1,3-dipolar trapping of these nitrones to homonuclear dipolarophiles is described. The new dipoles 8 reacted with N-methyl maleimide, generating diastereomeric adducts 14-16. In the reaction between 8 and dimethyl acetylenedicarboxylate, primary cycloadducts 17 and/or stable rearrangement products, azomethine ylides 18, are formed depending on the substitution pattern of the dipole. The structure of 18c is unambiguously assigned by X-ray crystallographic analysis. An X-ray crystal structure determination is also presented for the cyclopropylisoxazoloquinazoline 22 formed by a [3 + 2] addition of 8a to 21, the dimethyl acetylenedicarboxylate tetramer.     

Gln-Gly cleavage: Correlation between collision-induced dissociation and biological degradation

Tryptic digestion of the 150-residue human acidic salivary proline-rich protein 1 (PRP-1) generated eight peptides, two of which corresponded to the N-terminal 30-residue segment. In each of the other six tryptic peptides, a consensus repeat with the structure PQGPPQQGG was present. A facile Gln-Gly cleavage between the second and the third residues of the repeat was observed during collision-induced dissociation experiments. We postulate possible mechanisms to account for this reactivity, involving attack on the peptidyl carbonyl group by the Gln sidechain. Significantly, the Gln-Gly cleavage has been shown to be biologically important in the bacterial degradation of PRPs in saliva, generating bacteria-binding Pro-Gln C-termini. We suggest a link between the gas-phase chemistry and the biochemical degradation of these molecules.     

Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and lid bead chromatography

An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described. The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system. The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed. While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the ultrafiltration step increased the productivity of the aqueous two-phase system by a factor of more than 10. The thermoseparated water phase was then subjected to a polishing step using lid bead chromatography. Lid beads are a new type of restricted access chromatography beads, here with a positively charged inner core that adsorbed the remaining RNA while its inert surface layer prevented adsorption of the plasmid DNA thus passing in the flow-through of the column. Differently-sized plasmid DNA in the range of 2.7-20.5 kbp were also partitioned in the aqueous two-phase system. Within this size range, all plasmid DNA was exclusively extracted to the top phase. The complete process is free of additives and easy scalable for use in large scale production of plasmid DNA. The overall process yield for plasmid DNA was 69%.     

Ion spectroscopy: Where did it come from; where is it now; and where is it going?

The ASMS conference on ion spectroscopy brought together at Asilomar on October 16–20, 2009 a large group of mass spectrometrists working in the area of ion spectroscopy. In this introduction to the field, we provide a brief history, its current state, and where it is going. Ion spectroscopy of intermediate size molecules began with photoelectron spectroscopy in the 1960s, while electronic spectroscopy of ions using the photodissociation “action spectroscopic” mode became active in the next decade. These approaches remained for many years the main source of information about ionization energies, electronic states, and electronic transitions of ions. In recent years, high-resolution laser techniques coupled with pulsed field ionization and sample cooling in molecular beams have provided high precision ionization energies and vibrational frequencies of small to intermediate sized molecules, including a number of radicals. More recently, optical parametric oscillator (OPO) IR lasers and free electron lasers have been developed and employed to record the IR spectra of molecular ions in either molecular beams or ion traps. These results, in combination with theoretical ab initio molecular orbital (MO) methods, are providing unprecedented structural and energetic information about gas-phase ions.     

How the biomimetic assembly of membrane receptors into multivalent domains is regulated by a small ligand

In living cells, communication requires the action of membrane receptors that are activated following very small environmental changes. A binary all-or-nothing behavior follows, making the organism extremely efficient at responding to specific stimuli. Using a minimal system composed of lipid vesicles, chemical models of a membrane receptor and their ligands, we show that bio-mimetic ON/OFF assembly of high avidity, multivalent domains is triggered by small temperature changes. Moreover, the intensity of the ON signal at the onset of the switch is modulated by the presence of small, weakly binding divalent ligands, reminiscent of the action of primary messengers in biological systems. Based on the analysis of spectroscopic data, we develop a mathematical model that rigorously describes the temperature-dependent switching of the membrane receptor assembly and ligand binding. From this we derive an equation that predicts the intensity of the modulation of the ON signal by the ligand-messenger as a function of the pairwise binding parameters, the number of binding sites that it features and the concentration. The behavior of our system, and the model derived, highlight the usefulness of weakly binding ligands in the regulation of membrane receptors and the pitfalls inherent to their binding promiscuity, such as non-specific binding to the membrane. Our model, and the equations derived from it, offer a valuable tool for the study of membrane receptors in both biological and biomimetic settings. The latter can be exploited to program membrane receptor avidity on sensing vesicles, create hierarchical protocell tissues or develop highly specific drug delivery vehicles.

In lipid vesicles near their membrane phase-transition temperature, the presence of a small, weakly binding ligand tips the balance for the assembly of multivalent receptor domains. We recapitulate this behaviour using a global binding-clustering model.     

Application of comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry in human metabolomic studies

In this paper several characteristic features of time-of-flight mass spectrometry in coupling with gas chromatography are demonstrated and the parameters are compared to quadrupole mass analyzer. In the second part, the comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry was applied in human metabolomic field, particularly in the determination of pathological markers of Inherited Metabolic Disorders (IMDs).