Monitoring metabolites consumption and secretion in cultured cells using ultra-performance liquid chromatography quadrupole–time of flight mass spectrometry (UPLC–Q–ToF-MS)

Here we present an ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) method for extracellular measurements of known and unexpected metabolites in parallel. The method was developed by testing 86 metabolites, including amino acids, organic acids, sugars, purines, pyrimidines, vitamins, and nucleosides, that can be resolved by combining chromatographic and m/z dimensions. Subsequently, a targeted quantitative method was developed for 80 metabolites. The presented method combines a UPLC approach using hydrophilic interaction liquid chromatography (HILIC) and MS detection achieved by a hybrid quadrupole–time of flight (Q–ToF) mass spectrometer. The optimal setup was achieved by evaluating reproducibility and repeatability of the analytical platforms using pooled quality control samples to minimize the drift in instrumental performance over time. Then, the method was validated by analyzing extracellular metabolites from acute lymphoblastic leukemia cell lines (MOLT-4 and CCRF-CEM) treated with direct (A-769662) and indirect (AICAR) AMP activated kinase (AMPK) activators, monitoring uptake and secretion of the targeted compound over time. This analysis pointed towards a perturbed purine and pyrimidine catabolism upon AICAR treatment. Our data suggest that the method presented can be used for qualitative and quantitative analysis of extracellular metabolites and it is suitable for routine applications such as in vitro drug screening.     

Synthesis and characterisation of macromolecules containing multiple tetrazole functionalities

The synthesis of tetra-tetrazole macromolecules, containing various aromatic cores including benzene, pyridine and pyrazine directly attached to the tetrazole moieties, is described. This variation allowed for the generation of ligands with greater potential for metal ion complexation. Metal ion complexation reactions of the tetra-tetrazole macromolecules with the chelating pyridyl-tetrazole arms result in the formation of metal complexes where the metal ion was bound at the pendant arms rather than at the central core.     

Nucleon axial charge in full lattice QCD

The nucleon axial charge is calculated as a function of the pion mass in full QCD. Using domain wall valence quarks and improved staggered sea quarks, we present the first calculation with pion masses as light as 354 MeV and volumes as large as (3.5 fm)3. We show that finite volume effects are small for our volumes and that a constrained fit based on finite volume chiral perturbation theory agrees with experiment within 7% statistical errors.     

Time-resolved spectroscopic study of the reaction Cl + n-C5H12 --> HCl + C5H11 in solution

We study the hydrogen abstraction reaction from pentane by chlorine radicals using four different experimental approaches. We use two different solvents (CH2Cl2 and CCl4) and two different chlorine atom sources (photodissociation of dissolved Cl2 and two-photon photolysis of the solvent) to investigate their effects on the recombination and reactivity of the chlorine radical. All four experimental schemes involve direct probing of the transient chlorine population via a charge transfer transition with a solvent molecule. In one of the four approaches, photolysis of Cl2 in dichloromethane, we also monitor the nascent reaction products (HCl) by transient vibrational spectroscopy. Probing both the reactants and the products provides a comprehensive view of this bimolecular reaction in solution. Between one-third and two-thirds of the chlorine radicals that initially escape the solvent cage undergo diffusive geminate recombination with their partner radical (either another chlorine atom or the solvent radical). The rest react with pentane with the bimolecular rate constants k(bi) = (9.5 +/- 0.7) x 10(9) M(-1) s(-1) in CH2Cl2 and k(bi) = (7.4 +/- 2) x 10(9) M(-1) s(-1) in CCl4. The recombination yield phi(rec) depends on both the chlorine atom precursor and the solvent and is larger in the more viscous carbon tetrachloride solutions. The bimolecular reaction rate k(bi) depends only on the solvent and is consistent with a nearly diffusion-limited reaction.     

A synthesis of (+)-saxitoxin

An asymmetric synthesis of the bis-guanidinium poison, (+)-saxitoxin (STX), is described. Commencing from an N,O-acetal starting material made readily available through sulfamate ester C-H amination, the completed route to STX showcases the utility of oxathiazinane dioxide heterocycles for the assembly of polyfunctionalized amine derivatives. In the final preparative stages, an unusual nine-membered ring guanidine intermediate is oxidized selectively and made to undergo dehydrative cyclization to afford the tricyclic core of the natural product. Access to STX and related structures will provide unique pharmacological tools for the study of voltage-regulated Na+ ion channel proteins.     

Termolecular kinetics for the Mu + CO + M recombination reaction: A unique test of quantum rate theory

The room-temperature termolecular rate constants, k0, for the Mu + CO + M<==>MuCO + M (M = He, N2, Ar) recombination reaction have been measured by the muSR technique, and are reported for moderator gas pressures of up to approximately 200 bar (densities less, similar 0.4 x 10(22) molec cm(-3)). The experimental relaxation rates reveal an unusual signature, in being dominated by the electron spin-rotation interaction in the MuCO radical that is formed in the addition step. In N2 moderator, k0 = 1.2+/-0.1 x 10(-34) cm(6) s(-1), only about 30% higher than found in Ar or He. The experimental results are compared with theoretical calculations carried out on the Werner-Keller-Schinke (WKS) surface [Keller et al., J. Chem. Phys. 105, 4983 (1996)], within the framework of the isolated resonance model (IRM). The positions and lifetimes of resonance states are obtained by solving the complex Hamiltonian for the nonrotating MuCO system, using an L2 method, with an absorbing potential in the asymptotic region. Accurate values of the vibrational bound and resonance states of MuCO reveal unprecedented isotope effects in comparisons with HCO, due to the remarkable effect of replacing H by the very light Mu atom (m(Mu) approximately (1/9)m(H)). Due to its pronounced zero-point energy shift, there are only two (J = 0) bound states in MuCO. Contributions from nonzero J states to the termolecular rate constants are evaluated through the J-shifting approximation, with rotational constants evaluated at the potential minimum. The value of the important A constant (181 cm(-1)) used in this approximation was supported by accurate J = K = 1 calculations, from which A = 180 cm(-1) was obtained by numerical evaluation. The calculations presented here, with a "weak collision factor" beta c = 0.001, indicative of the very sparse density of MuCO states, give a very good account of both the magnitude and pressure dependence of the experimental rates, but only when the fact that the two initially bound (J = 0) states become resonances for J > 0 is taken into account. This is the first time in IRM calculations of atom-molecule recombination reactions where J not equal to 0 states have proven to be so important, thus providing a truly unique test of quantum rate theory.     

Vibrationally mediated photodissociation of ammonia: the influence of N-H stretching vibrations on passage through conical intersections

Velocity map ion imaging of the H atoms formed in the photodissociation of vibrationally excited ammonia molecules measures the extent of adiabatic and nonadiabatic dissociation for different vibrations in the electronically excited state. Decomposition of molecules with an excited symmetric N-H stretch produces primarily ground state NH(2) along with a H atom. The kinetic energy release distribution is qualitatively similar to the ones from dissociation of ammonia excited to the electronic origin or to several different levels of the bending vibration and umbrella vibration. The situation is very different for electronically excited molecules containing a quantum of antisymmetric N-H stretch. Decomposition from that state produces almost solely electronically excited NH(2)*, avoiding the conical intersection between the excited state and ground state surfaces. These rotationally resolved measurements agree with our previous inferences from lower resolution Doppler profile measurements. The production of NH(2)* suggests that the antisymmetric stretching excitation in the electronically excited molecule carries it away from the conical intersection that other vibrational states access.     

Theoretical study of the syn and anti thiophene-2-aldehyde conformers using density functional theory and normal coordinate analysis

An extensive computational study of thiophene-2-aldehyde conformers syn and anti has been carried out using density functional (DFT). From these calculations, B3LYP/6-31G(d) has been chosen as it produces results remarkably close in comparison with experimental ones, with less demanding computational time. Data obtained from DFT computation were used to perform a normal coordinate analysis to complement and give insight in the experimental vibrational assignment. Calculated dipole moments and relative stabilities of isomers coherently support experimental statements given in the literature.     

Two-dimensional optical three-pulse photon echo spectroscopy. I. Nonperturbative approach to the calculation of spectra

The nonperturbative approach to the calculation of nonlinear optical spectra of Seidner et al. [J. Chem. Phys. 103, 3998 (1995)] is extended to describe four-wave mixing experiments. The system-field interaction is treated nonperturbatively in the semiclassical dipole approximation, enabling a calculation of third order nonlinear spectroscopic signals directly from molecular dynamics and an efficient modeling of multilevel systems exhibiting relaxation and transfer phenomena. The method, coupled with the treatment of dynamics within the Bloch model, is illustrated by calculations of the two-dimensional three-pulse photon echo spectra of a simple model system-a two-electronic-level molecule. The nonperturbative calculations reproduce well-known results obtained by perturbative methods. Technical limitations of the nonperturbative approach in dealing with a dynamic inhomogeneity are discussed, and possible solutions are suggested. An application of the approach to an excitonically coupled dimer system with emphasis on the manifestation of complex exciton dynamics in two-dimensional optical spectra is presented in paper II Pisliakov et al. [J. Chem. Phys. 124, 234505 (2006), following paper].