Synthesis of (−)-Piperitylmagnolol Featuring <i>ortho</i>-Selective Deiodination and Pd-Catalyzed Allylation

A 1,4-addition strategy using an enone and a copper reagent was studied for the synthesis of (−)-piperitylmagnolol. A MOM-protected biphenol copper reagent was added to BF₃·OEt₂-activated 4-isopropyl­cyclo­hexenone, whereas 1,4-addition of protected monophenol reagents possessing an allyl group was found to be unsuccessful. The allyl group was later attached to the <i>p</i>-,<i>p</i>′-diiodo-biphenol ring by Pd-catalyzed coupling with allylborate. The aforementioned iodide was synthesized using a new method for <i>ortho</i>-selective deiodination of <i>o</i>-,<i>p</i>-diiodophenols

A QM/MM study of nitric oxide reductase-catalysed N₂O formation

<div><p>Nitrous oxide (N₂O), with a greenhouse effect 300 times that of CO₂, is increasingly eliminated into the atmosphere. Using a hybrid quantum mechanics/molecular mechanics (QM/MM) method, we examined nitric oxide reductase-catalysed N₂O formation, which includes two important chemical reactions of N–N bond formation and N–O bond cleavage. The N–N bond formation has no activation barrier, but N–O bond cleavage exhibits an activation barrier of 20.9 kcal·mol⁻¹ at the QM/MM level. We show that the N–O bond cleavage occurs via a hyponitrous intermediate (Fe_B (II; <i>s</i> = 4/2)/N₂O₂ (−1; <i>s</i> = 1/2)/ (III; <i>s</i> = −1/2)), with bidentate coordination between Glu211 and a non-heme iron atom. The Glu211 coordination decreases the N–O bond cleavage energy barrier by inhibiting the formation of stable, five-membered ring intermediate (Fe_B–O¹–N¹–N²–O²–).</p></div

Substrate-mediated proton relay mechanism for the religation reaction in topoisomerase II

<div><p>The DNA religation reaction of yeast type II topoisomerase (topo II) was investigated to elucidate its metal-dependent general acid/base catalysis. Quantum mechanical/molecular mechanical calculations were performed for the topo II religation reaction, and the proton transfer pathway was examined. We found a substrate-mediated proton transfer of the topo II religation reaction, which involves the 3′ OH nucleophile, the reactive phosphate, water, Arg781, and Tyr782. Metal A stabilizes the transition states, which is consistent with a two-metal mechanism in topo II. This pathway may be required for the cleavage/religation reaction of topo IA and II and will provide a general explanation for the catalytic mechanism in the topo IA and II.</p></div

A QM/MM Study of the l‑Threonine Formation Reaction of Threonine Synthase: Implications into the Mechanism of the Reaction Specificity

Threonine synthase catalyzes the most complex reaction among the pyridoxal-5′-phosphate (PLP)-dependent enzymes. The important step is the addition of a water molecule to the Cβ–Cα double bond of the PLP−α-aminocrotonate aldimine intermediate. Transaldimination of this intermediate with Lys61 as a side reaction to form α-ketobutyrate competes with the normal addition reaction. We previously found that the phosphate ion released from the <i>O</i>-phospho-l-homoserine substrate plays a critical role in specifically promoting the normal reaction. In order to elucidate the detailed mechanism of this “product-assisted catalysis”, we performed comparative QM/MM calculations with an exhaustive search for the lowest-energy-barrier reaction pathways starting from PLP−α-aminocrotonate aldimine intermediate. Satisfactory agreements with the experiment were obtained for the free energy profile and the UV/vis spectra when the PLP pyridine N1 was unprotonated and the phosphate ion was monoprotonated. Contrary to an earlier proposal, the base that abstracts a proton from the attacking water was the ε-amino group of Lys61 rather than the phosphate ion. Nevertheless, the phosphate ion is important for stabilizing the transition state of the normal transaldimination to form l-threonine by making a hydrogen bond with the hydroxy group of the l-threonine moiety. The absence of this interaction may account for the higher energy barrier of the side reaction, and explains the mechanism of the reaction specificity afforded by the phosphate ion product. Additionally, a new mechanism, in which a proton temporarily resides at the phenolate O3′ of PLP, was proposed for the transaldimination process, a prerequisite step for the catalysis of all the PLP enzymes

Pedigrees of six Japanese familial episodic pain syndrome in Japanese families.

<p>(A) Some ^a)Family 2 and ^b)Family 3 members have been reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154827#pone.0154827.ref013" target="_blank">13</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154827#pone.0154827.ref014" target="_blank">14</a>]. Black and white symbols indicate affected and unaffected individuals, respectively. Gray symbols indicate individuals with unknown phenotypic status. Squares and circles indicate males and females, respectively. Slashes indicate deceased individuals. “P” indicates probands. Blue arrows indicate exome sequenced individuals. * indicates linkage analysis performed individuals. The genotype of <i>SCN11A</i> p.R222H (Family 1, 2, 4, 5 and 6) or <i>SCN11A</i> p.R222S (Family 3) for each individual is illustrated. (B) Sequence chromatography of the identified <i>SCN11A</i> mutations.</p

Three subtypes of clinical manifestations.

<p>Three subtypes of clinical manifestations.</p

Exome analysis filtering process in the three Japanese familial episodic pain syndrome families.

<p>Exome analysis was performed for three affected members in Family 1, four affected and four unaffected members in Family 2, and two affected and one unaffected member in Family 3. Exome data was processed through seven filtering steps: (1) non-synonymous, (2) read depth ≥ 8, (3) not registered in dbSNP135, (4) MAF < 0.01 in Japanese patients from 1000 Genomes database, (5) heterozygote in affected members and not present in unaffected members, (6) located on 3p22 linkage region, and (7) variants in the same gene among all three families. Numbers in boxes represent the numbers of variants after each filtering step.</p

Genome-wide linkage analysis in two Japanese familial episodic pain syndrome families.

<p>Genome-wide linkage analysis was performed for eight affected and five unaffected members in Family 1, and four affected and four unaffected members in Family 2. Parametric linkage analysis was performed using 386 genetic markers (including 382 microsatellite genetic markers) that were 10 cM apart and covered 22 autosomes, as well as additional SNP markers. GeneHunter software was used.</p

Characteristics of Nav1.9-overexpressing ND7/23 cells.

<p>(A) The typical traces of Na⁺ currents under 3 μM TTX treatment among Nav1.9-overexpressing ND7/23 cells (Control, WT, R222S, and R222H) were selected at step pulses from −80–0 mV for 100 ms with 20-mV increments for clarity. Control indicates ND7/23 cells without Nav1.9 transfection. The data were obtained at 28°C. Red-colored traces were obtained at −20 mV step pulse. (B) Current-voltage relationships for each Nav1.9-overexpressing ND7/23 cell (Control <i>n</i> = 5, WT <i>n</i> = 5, R222S <i>n</i> = 5, and R222H <i>n</i> = 4). Step pulses were applied from −120–30 mV for 100 ms in 10-mV increments. (C) Comparison of activation of each Nav1.9-overexpressing ND7/23 cell. The Boltzmann fit correspond to V_1/2 (WT: −44.83 ± 2.44 mV, <i>n</i> = 5, R222S: −39.5 ± 2.09 mV, <i>n</i> = 5, R222H: −42.22 ± 2.91 mV, <i>n</i> = 4).</p

R222S mutation increases excitability in DRG neurons.

<p>(A) Small DRG neuron (< 25μm) responses to 500-ms depolarizing current steps of 10, 110, and 210 pA in WT and R222S mice. The parameter of the first AP obtained during current injections of 210 pA, showing calculated maximum rate of rise (B) and fall (C) of AP firing. Open and closed circles represent WT (<i>n</i> = 4) and R222S mice (<i>n</i> = 5), respectively. (D) Input impedance was measured at an injection current of 10 pA. Open and closed columns represent WT (<i>n</i> = 6) and R222S mice (<i>n</i> = 5), respectively. (E) Comparison of repetitive action potentials between WT and R222S mice. Open and closed circles represent WT (<i>n</i> = 6) and R222S mice (<i>n</i> = 5), respectively. The range of 500-ms-step current injections was 10–210 pA. Data are presented as mean ± S.E.M. (*<i>p</i> < 0.05; two-sided Student’s <i>t</i> test).</p