Ring-Expansion Metathesis Polymerization: Catalyst-Dependent Polymerization Profiles

Ring-expansion metathesis polymerization (REMP) mediated by recently developed cyclic Ru catalysts has been studied in detail with a focus on the polymer products obtained under varied reaction conditions and catalyst architectures. Depending upon the nature of the catalyst structure, two distinct molecular weight evolutions were observed. Polymerization conducted with catalysts bearing six-carbon tethers displayed rapid polymer molecular weight growth which reached a maximum value at ca. 70% monomer conversion, resembling a chain-growth polymerization mechanism. In contrast, five-carbon-tethered catalysts led to molecular weight growth that resembled a step-growth mechanism with a steep increase occurring only after 95% monomer conversion. The underlying reason for these mechanistic differences appeared to be ready release of five-carbon-tethered catalysts from growing polymer rings, which competed significantly with propagation. Owing to reversible chain transfer and the lack of end groups in REMP, the final molecular weights of cyclic polymers was controlled by thermodynamic equilibria. Large ring sizes in the range of 60−120 kDa were observed at equilibrium for polycyclooctene and polycyclododecatriene, which were found to be independent of catalyst structure and initial monomer/catalyst ratio. While six-carbon-tethered catalysts were slowly incorporated into the formed cyclic polymer, the incorporation of five-carbon-tethered catalysts was minimal, as revealed by ICP-MS. Further polymer analysis was conducted using melt-state magic-angle spinning ^(13)C NMR spectroscopy of both linear and cyclic polymers, which revealed little or no chain ends for the latter topology

Biased small-molecule ligands for selective inhibition of HIV-1 cell entry via CCR5

Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small‐molecule receptor antagonists for inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small‐molecule CCR5 agonists as HIV‐1 fusion inhibitors. A virus‐free cell‐based fusion reporter assay, based on mixing “effector cells” (expressing HIV Env and luciferase activator) with “target cells” (expressing CD4, CCR5 wild type or a selection of well‐described mutations, and luciferase reporter), was used as fusion readout. Receptor expression was evaluated by ELISA and fluorescence microscopy. On CCR5 WT, Maraviroc and Aplaviroc inhibited fusion with high potencies (EC (50) values of 91 and 501 nM, respectively), whereas removal of key residues for both antagonists (Glu283Ala) or Maraviroc alone (Tyr251Ala) prevented fusion inhibition, establishing this assay as suitable for screening of HIV entry inhibitors. Both ligands inhibited HIV fusion on signaling‐deficient CCR5 mutations (Tyr244Ala and Trp248Ala). Moreover, the steric hindrance CCR5 mutation (Gly286Phe) impaired fusion, presumably by a direct hindrance of gp120 interaction. Finally, the efficacy switch mutation (Leu203Phe) – converting small‐molecule antagonists/inverse agonists to full agonists biased toward G‐protein activation – uncovered that also small‐molecule agonists can function as direct HIV‐1 cell entry inhibitors. Importantly, no agonist‐induced receptor internalization was observed for this mutation. Our studies of the pharmacodynamic requirements for HIV‐1 fusion inhibitors highlight the possibility of future development of biased ligands with selective targeting of the HIV–CCR5 interaction without interfering with the normal functionality of CCR5

Anisotropic chemical shifts and spin rotation constants of 15N from liquid and solid state NMR: Nitrobenzene

The study of the 15N spin-lattice relaxation time T1 in nitrobenzene at 14 and 32 MHz from −10 to 60°C showed that at 32 MHz relaxation due to anisotropic chemical shift is predominant. At low frequencies, the relaxation is caused mainly by spin rotation and at low temperature also by intermolecular dipole-dipole interaction. From the powder spectrum in solid nitrobenzene, the principal elements of the shielding tensor σ were obtained: σxx = −273 ± 10 ppm, σyy = +94 ± 10 ppm, and σzz = +156 ± 10 ppm relative to liquid nitrobenzene, Δσ = σ xx - ½(σ yy + σ zz) = -398 ± 20ppm. From the almost axially symmetric σ -tensor, the spin rotation constants were calculated: C‖ = 11.4 ± 1.5 kHz and C⊥ = 1.35 ± 0.5 kHz, where C‖ is the component parallel to the twofold axis of the molecule. These values for Δσ and the spin rotation constants are in excellent agreement with those obtained by analysis of the relaxation data. A comparison of anisotropic chemical shifts and spin rotation constants for 15N and 13C in isoelectronic compounds is given

Spin echo formation in the presence of stochastic dynamics

Spin echo formation in magnetic field gradients in the presence of fast stochastic motion is studied for hyperpolarized He3 gas at different diffusivities. The fast translational motion leads to frequency shifts already during echo formation, which can be described analytically for a linear gradient. Despite complete signal loss at the position of the spin echo itself, considerable intensity can be preserved at an earlier time (2τ rather than 2τ, where τ is the pulse delay). Hence, the phenomenon is designated as a pseudo spin echo. © 2007 The American Physical Society.Fil: Zänker, Paul P.. Max Planck Institute For Polymer Research; AlemaniaFil: Schmidt, Jochen. Max Planck Institute For Polymer Research; AlemaniaFil: Schmiedeskamp, Jörg. Max Planck Institute For Polymer Research; AlemaniaFil: Acosta, Rodolfo Héctor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Física Enrique Gaviola. Universidad Nacional de Córdoba. Instituto de Física Enrique Gaviola; ArgentinaFil: Spiess, Hans W.. Max Planck Institute For Polymer Research; Alemani

Spin echo experiments on 13C, 2H, 1H, and 19F in some small molecules in the liquid phase

The dependence on the π pulse repetition rate (2τ)−1 of the Carr-Purcell (CP) spin echo decay constant R is studied for four nuclei in C6H6, C6D6, C6F6, C6H12, C6H5CH3, CH3I, H2O, D2O, and CS2. Both deuteron resonances, the proton resonances of CH3I, of extremely pure H2O and C6H6 and the 13C resonance of CS2 yield straight lines when R is plotted vs. (2τ)2, i.e., R is governed by transverse relaxation and diffusion. However, in some unexpected cases, T2 is found to be smaller than T1. The H and F resonances of C6H6, C6F6, and H2O with traces of impurities do not give straight-line plots of R vs. (2τ)2. An oscillatory dependence of R on the pulse repetition rate is found for the 13C resonances of C6H6 and C6H5CH3. It can be shown to be due to the J coupling of the 13C spins to the directly bonded protons. The theory developed for exchange of chemically shifted spins can be applied and is extended for the slow exchange limit to an AX3 system in an effort to explain the results of methyl 13C quantitatively. Because of the sensitivity of CP measurements on instrumental effects a detailed description is given of the measurement procedures and of the equipment of which a superconducting solenoid is an essential part. A connection between Carr-Purcell measurements, of the Gill-Meiboom version, and spin-locking experiments is pointed out

Molecular motion in liquid toluene from a study of 13C and 2D relaxation times

The 13C nuclear spin-lattice relaxation times for ring and methyl carbons in liquid toluene were studied from −95°C to +60°C at frequencies of 14 and 61 MHz. Data were taken for protonated as well as deuterated toluene. The results were analyzed in terms of three relaxation mechanisms: intramolecular dipole-dipole coupling, spin-rotation interaction, and anisotropic chemical shift. The last mechanism gives a significant contribution only to the relaxation rate of the ring carbons of the deuterated species at 61 MHz and low temperatures. A tentative value of Δσ = 295 ppm is obtained in this case. In order to separate the contributions of the dipole-dipole and spin-rotation interactions the 13C data are compared with deuteron relaxation times. Comparison of the 13C data in the protonated and deuterated form of toluene shows that the correlation times for the ring differ by 20% and an even larger effect of isotopic substitution is found for the methyl group. It is demonstrated that the fast internal motion of the methyl group cannot be studied quantitatively using deuteron or 13C intramolecular dipole-dipole relaxation rates alone because of the sensitivity of the results to the angle, varpi, the Z-axis of the electric field gradient, or the internuclear vector, respectively, forms with the C3 axis. Analysis of the relaxation rates due to spin-rotation interaction yields τj (internal), the correlation time of angular momentum of the internal motion directly. The correlation time of reorientation τc (internal) is calculated from τj (internal) using Gordon's extended diffusion model which is applied to a symmetric rotor with a fixed axis. It is found that both τj (internal) and τc (internal) are of the same magnitude as the correlation time of the free rotor. The ratio of correlation times of the overall and internal reorientation ranges from approximately 200 at the melting point to approximately 13 at +60°

How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis

<p>Abstract</p> <p>Background</p> <p>In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus <it>Trichoderma reesei</it>. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis.</p> <p>Results</p> <p>After expression in the yeast <it>Kluyveromyces lactis</it>, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from <it>T. reesei</it>. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin-induced reduction in particle size and crystallinity resulted in high cellulose hydrolysis rates.</p> <p>Conclusions</p> <p>Recombinant swollenin can be easily produced with the robust yeast <it>K. lactis</it>. Moreover, swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects different cellulosic substrates and their hydrolysis.</p

Spin rotation interaction and anisotropic chemical shift in 13CS2

The 13C nuclear spin-lattice relaxation time T1 was studied in liquid CS2 from -106°C to +35°C at resonance frequencies of 14, 30, and 62 MHz. The relaxation is caused by anisotropic chemical shift and spin-rotation interaction. It is shown that for linear molecules the spin-rotation constant C and the anisotropy of the chemical shift &#916;&#963; can be obtained from the relaxation rates without use of adjustable parameters. The analysis yields: C = -13.8 ± 1.4 kHz and &#916;&#963; = 438 ± 44 ppm

"Hook"-calibration of GeneChip-microarrays: Chip characteristics and expression measures

<p>Abstract</p> <p>Background</p> <p>Microarray experiments rely on several critical steps that may introduce biases and uncertainty in downstream analyses. These steps include mRNA sample extraction, amplification and labelling, hybridization, and scanning causing chip-specific systematic variations on the raw intensity level. Also the chosen array-type and the up-to-dateness of the genomic information probed on the chip affect the quality of the expression measures. In the accompanying publication we presented theory and algorithm of the so-called hook method which aims at correcting expression data for systematic biases using a series of new chip characteristics.</p> <p>Results</p> <p>In this publication we summarize the essential chip characteristics provided by this method, analyze special benchmark experiments to estimate transcript related expression measures and illustrate the potency of the method to detect and to quantify the quality of a particular hybridization. It is shown that our single-chip approach provides expression measures responding linearly on changes of the transcript concentration over three orders of magnitude. In addition, the method calculates a detection call judging the relation between the signal and the detection limit of the particular measurement. The performance of the method in the context of different chip generations and probe set assignments is illustrated. The hook method characterizes the RNA-quality in terms of the 3'/5'-amplification bias and the sample-specific calling rate. We show that the proper judgement of these effects requires the disentanglement of non-specific and specific hybridization which, otherwise, can lead to misinterpretations of expression changes. The consequences of modifying probe/target interactions by either changing the labelling protocol or by substituting RNA by DNA targets are demonstrated.</p> <p>Conclusion</p> <p>The single-chip based hook-method provides accurate expression estimates and chip-summary characteristics using the natural metrics given by the hybridization reaction with the potency to develop new standards for microarray quality control and calibration.</p