Termination Mechanism in the Radical Polymerization of Methyl Methacrylate and Styrene Determined by the Reaction of Structurally Well-Defined Polymer End Radicals

A novel method to determine the termination mechanism of radical polymerization, i.e., the selectivity between disproportionation (Disp) and combination (Comb), is developed. The method relies on product analyses of the reaction of polymer-end radicals, which are generated from structurally well-controlled living polymers, and the analyses of molecular weight and end-group structure of the product polymers by GPC, mass spectroscopy, and ¹H NMR unambiguously determined the contribution of two competing pathways. The termination mechanism in the polymerization of methyl methacrylate (MMA) and styrene was investigated as a proof of principle of the method by using the corresponding polymers prepared by organotellurium-mediated radical polymerization. The ratios of Disp and Comb (<i>D</i>/<i>C</i>) of poly­(methyl methacrylate) (PMMA) or polystyrene (PSt) end radicals at 25 °C were 73/27 or 15/85, respectively, and the results agreed well with the previous reports. The contribution of the Comb increased at higher temperature in both cases, though the temperature dependence was less pronounced in PSt radicals (<i>D</i>/<i>C</i> = 67/37 and 13/87 at 100 °C for PMMA and PSt, respectively). Thermodynamic parameters were determined as ΔΔ<i>G</i>^‡_d/c = (−6.9 ± 0.3) – <i>T</i> × (−14.4 ± 1.0) × 10^–3 (kJ mol^–1) for PMMA and ΔΔ<i>G</i>^‡_d/c = (−2.0 ± 0.5) – <i>T</i> × (−20.8 ± 1.5) × 10^–3 (kJ mol^–1) for PSt, in which ΔΔ<i>G</i>^‡_d/c and <i>T</i> are difference in Gibbs energy undergoing Disp and Comb, and temperature in Kelvin, respectively, by carrying out the same experiments between −20 to +100 °C. The parameters reveal that Comb is enthalpically less favored but entropically more favored than Disp in both cases. The effects of molecular weight (chain length) were also investigated, and the <i>D</i>/<i>C</i> ratio became constant when the molecular weight of polymers was more than about 3000

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Bright Fluorescence Monitoring System Utilizing<i>Zoanthus</i> sp. Green Fluorescent Protein (<i>ZsGreen</i>) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

<div><p>G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer’s disease and Parkinson’s disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).</p> </div

Mechanism of Cu(I)/Cu(0)-Mediated Reductive Coupling Reactions of Bromine-Terminated Polyacrylates, Polymethacrylates, and Polystyrene

The mechanism of the Cu­(I)/Cu(0)-mediated reductive coupling reactions of bromine-terminated polymer chain-end radicals, so-called atom-transfer radical coupling (ATRC), is studied. Poly­(methyl acrylate) (PMA), poly­(methyl methacrylate) (PMMA), and polystyrene (PSt), prepared by atom-transfer radical polymerization (ATRP), were activated by an excess amount of Cu­(I)Br and Cu(0) in the presence of tris­[2-(dimethylamino)­ethyl]­amine (Me₆TREN), and the structural analyses of the resulting polymer products and deuterium-labeling experiments unambiguously determined the mechanism. While PMMA and PSt reacted by a radical mechanism as expected, PMA-bromide was reduced to an anionic species, which was most likely an organocopper species. Trapping experiments with TEMPO suggested that the polymer chain-end radicals were generated in all cases by the reduction of the bromine-terminated polymers by low-valent Cu species. However, the PMA chain-end radical was further reduced to the anionic species from which coupling products formed in low yield

Quantitative β-galactosidase assays for homo- and hetero-dimerization between human-GPCRs in NMY63 strain.

<p>NMY63 yeast strain was transformed with GPCR-Nub<i>G</i> indicated at the left and SSTR2-Cub-LexA-VP16 (A), ADRB2-Cub-LexA-VP16 (B), or HTR1A-Cub-LexA-VP16 (C). The control prey plasmid was pPR3-C mock vector. Error bars represent the standard deviations (<i>n = </i>3).</p

Detection for dimerization and ligand-induced conformational changes of human GPCRs.

<p>(A) Quantitative β-galactosidase assays for heterodimerization of AGTR1 in NMY63 strain. NMY63 yeast strain was transformed with GPCR-Nub<i>G</i> indicated at the bottom and AGTR1-Cub-LexA-VP16. (B–D) Ligand assays for detection of conformational changes in GPCR dimerizations. (B) AT₁/AT₂ (AGTR1/AGTR2) heterodimers. Incubation time, 18 h. Angiotensin II conc., 0 or 10 µM. (C) MT₁/MT₂ (MTNR1A/MTNR1B) heterodimers. Incubation time, 18 h. Melatonin conc., 0 or 10 µM. (D) GABA_B1a/GABA_B2 (GABBR1a/GABBR2) heterodimers. Incubation time, 18 h. GABA conc., 0 or 100 µM. The control prey plasmid was pPR3-C mock vector. Error bars represent the standard deviations (<i>n = </i>3). (*<i>P</i><0.05).</p

Rapid, Facile Detection of Heterodimer Partners for Target Human G-Protein-Coupled Receptors Using a Modified Split-Ubiquitin Membrane Yeast Two-Hybrid System

<div><p>Potentially immeasurable heterodimer combinations of human G-protein-coupled receptors (GPCRs) result in a great deal of physiological diversity and provide a new opportunity for drug discovery. However, due to the existence of numerous combinations, the sets of GPCR dimers are almost entirely unknown and thus their dominant roles are still poorly understood. Thus, the identification of GPCR dimer pairs has been a major challenge. Here, we established a specialized method to screen potential heterodimer partners of human GPCRs based on the split-ubiquitin membrane yeast two-hybrid system. We demonstrate that the mitogen-activated protein kinase (MAPK) signal-independent method can detect ligand-induced conformational changes and rapidly identify heterodimer partners for target GPCRs. Our data present the abilities to apply for the intermolecular mapping of interactions among GPCRs and to uncover potential GPCR targets for the development of new therapeutic agents.</p></div

Detection for dimerization of yeast Ste2p deletion mutants (TM1–5 and TM6–7) in NMY62 strain.

<p>(A) Schematic of Ste2ΔC and the deletion mutants. Transmembrane (TM) domains are indicated with pillar-type boxes, and the Cub (Cub-LexA-VP16) or Nub<i>G</i> (Nub with I13G mutation) is depicted as a rounded rectangle. (B) Growth assay without α-factor (<i>left panels</i>) and with 5 µM α-factor (<i>right panels</i>). Each cell was spotted in serial 10-fold dilutions on SD –Leu, Trp, Ade and His dropout plate. (C) Quantitative β-galactosidase activity in yeast cells containing various combinations of plasmids. Error bars represent the standard deviations (<i>n = </i>3). The control prey plasmids were pPR3-C mock vector (empty vector) and pPR3-HXT1.</p

Screening of candidate heterodimer partners of AT₁ angiotensin receptor (AGTR1).

<p>(A) Workflow of a yeast two-hybrid screen. Prey library was transformed into the NMY63 yeast strains harboring AGTR1 bait vector, and the selection with growth reporter genes was performed. Following isolation of prey plasmids from each colony, the obtained GPCR clones were determined by sequencing analysis. (B) Quantitative β-galactosidase assays for homo- and hetero-dimerization of AGTR1 in NMY63 strain. NMY63 yeast strain was transformed with GPCR-Nub<i>G</i> indicated at the left and AGTR1-Cub-LexA-VP16. The control prey plasmid was pPR3-C mock vector. Error bars represent the standard deviations (<i>n = </i>3).</p

Activation of human neurotensin receptor subtype-1 (hNTSR1) by membrane-tethered neurotensin.

<p>(<i>A</i>) Amino acid sequences of membrane-tethered peptides. (<i>B</i>, <i>C</i>) Yeast strain IMFD-72ZsD, which coexpresses pGK421-NTSR1 and either pGK426-NTS42 (NTS), pGK426-NTS(8-13)42 (NTS(8-13)), pGK426-NMN42 (NMN) or pGK426-alpha42 (α-factor), was incubated in pH-adjusted SD selective medium. (<i>B</i>) The GFP fluorescence of 10,000 cells was measured by flow cytometry. Mean values of the green fluorescence signal of 10,000 cells. Error bars represent the standard deviations (<i>n</i> = 3); *, <i>p</i> < 0.05, and ***, <i>p</i> < 0.001, by one-way ANOVA, Tukey’s post test. (<i>C</i>) Fluorescence microscopy analysis of the cells incubated for 24 h. Cells were examined using the 40× objective lens of a fluorescence microscope. Exposure time was 0.67 s. The left photographs are fluorescence micrographs, and the right photographs are bright-field micrographs. </p