Additional file 1: of Tubulin is a molecular target of the Wnt-activating chemical probe

Supplemental results of cell-based assays and tubulin polymerization assay. Figure S1. Measurements of fold changes in the cellular area. Figure S2. Inhibitory activity on the tubulin polymerization and the cellular microtubule network. Figure S3. The absorbance and fluorescence profiles of AMBMP. Figure S4. Measurements of growth inhibitory activity, cell cycle distribution, and mitotic spindle of MDA-MB231 cells treated with AMBMP. (DOCX 6384 kb

C(sp³)–H Alkenylation Catalyzed by Cationic Alkylhafnium Complexes: Stereoselective Synthesis of Trisubstituted Alkenes from 2,6-Dimethylpyridines and Internal Alkynes

Dibenzylhafnium complexes <b>3a</b>–<b>d</b>, supported by dianionic bidentate or tridentate ligands, upon activation via abstraction by either [Ph₃C]­[B­(C₆F₅)₄] or B­(C₆F₅)₃ served as catalysts for the C­(sp³)–H alkenylation of 2,6-dimethylpyridines with dialkylalkynes to give corresponding C­(sp³)–H alkenylated products <b>6</b>. Complex <b>3c</b>, containing a pyridine arm in the ligand skeleton, exhibited the highest catalytic activity among <b>3a</b>–<b>d</b>; initial addition of 2,6-dimethylpyridine (<b>4a</b>) to the C₆D₅Br solution of <b>3c</b> followed by [Ph₃C]­[B­(C₆F₅)₄] and 3-hexyne (<b>5a</b>) produced trisubstituted alkene <b>6aa</b> in stereoselective manner in up to 50% yield without any byproducts, while the addition of <b>5a</b> prior to <b>4a</b> and [Ph₃C]­[B­(C₆F₅)₄] to the C₆D₅Br solution of <b>3c</b> generated <b>6aa</b>, together with the formation of byproduct (<i>E</i>)-(2-ethylpent-2-en-1-yl)­benzene (<b>7</b>). When an asymmetrical pyridine, 3-bromo-2,6-dimethylpyridine, was used as the coupling partner, the corresponding trisubstituted alkene was obtained selectively. Catalytically active cationic benzylhafnium complexes <b>8a</b>–<b>d</b>, which were prepared by the reactions of <b>3a</b>–<b>d</b> and B­(C₆F₅)₃, respectively, were characterized by ¹H, ¹³C, and ¹⁹F NMR spectroscopy. Kinetic studies of the catalytic reaction between <b>4a</b> and 4-octyne (<b>5b</b>) using <b>3c</b> and [Ph₃C]­[B­(C₆F₅)₄] in C₆D₅Br revealed that the catalytic reaction was zero-order for both <b>4a</b> and <b>5b</b>, indicating that the rate-determining step involved the C­(sp³)–H bond activation of <b>4a</b> by vinylhafnium intermediate <b>11c</b>

Preparation and Structure of Iminopyrrolyl and Amidopyrrolyl Complexes of Group 2 Metals

Reactions of <i>N</i>-aryliminopyrrolyl ligand <b>1a</b>, 2-(2,6-^<i>i</i>Pr₂C₆H₃NCH)-C₄H₃NH (Imp^Dipp-H), with dibenzylcalcium gave two types of pyrrolylcalcium complexes, bis­(iminopyrrolyl)calcium (<b>2a</b>) and (amidopyrrolyl)­calcium (<b>3a</b>), via alkane elimination and ligand alkylation reaction, respectively. Preparation of a mono­(iminopyrrolyl) complex, (iminopyrrolyl)­Ca­[N­(SiMe₃)₂]­(THF)₂ (<b>4a</b>), was accomplished by the addition of 1 equiv of <b>1a</b> to Ca­[N­(SiMe₃)₂]₂(THF)₂. A series of group 2 metal bis­(iminopyrrolyl) complexes, [(Imp^Dipp)₂M­(THF)₃] (M = Sr (<b>5a</b>), Ba, (<b>6a</b>)) and [(Imp^Me)₂Ca­(THF)₂] (<b>2b</b>) (2-(4-MeC₆H₄NCCH₃)-C₄H₃NH (Imp^Me-H)), was selectively prepared via amine elimination reactions, and their molecular structures were clarified by X-ray diffraction studies

C(sp³)–H Alkenylation Catalyzed by Cationic Alkylhafnium Complexes: Stereoselective Synthesis of Trisubstituted Alkenes from 2,6-Dimethylpyridines and Internal Alkynes

Dibenzylhafnium complexes <b>3a</b>–<b>d</b>, supported by dianionic bidentate or tridentate ligands, upon activation via abstraction by either [Ph₃C]­[B­(C₆F₅)₄] or B­(C₆F₅)₃ served as catalysts for the C­(sp³)–H alkenylation of 2,6-dimethylpyridines with dialkylalkynes to give corresponding C­(sp³)–H alkenylated products <b>6</b>. Complex <b>3c</b>, containing a pyridine arm in the ligand skeleton, exhibited the highest catalytic activity among <b>3a</b>–<b>d</b>; initial addition of 2,6-dimethylpyridine (<b>4a</b>) to the C₆D₅Br solution of <b>3c</b> followed by [Ph₃C]­[B­(C₆F₅)₄] and 3-hexyne (<b>5a</b>) produced trisubstituted alkene <b>6aa</b> in stereoselective manner in up to 50% yield without any byproducts, while the addition of <b>5a</b> prior to <b>4a</b> and [Ph₃C]­[B­(C₆F₅)₄] to the C₆D₅Br solution of <b>3c</b> generated <b>6aa</b>, together with the formation of byproduct (<i>E</i>)-(2-ethylpent-2-en-1-yl)­benzene (<b>7</b>). When an asymmetrical pyridine, 3-bromo-2,6-dimethylpyridine, was used as the coupling partner, the corresponding trisubstituted alkene was obtained selectively. Catalytically active cationic benzylhafnium complexes <b>8a</b>–<b>d</b>, which were prepared by the reactions of <b>3a</b>–<b>d</b> and B­(C₆F₅)₃, respectively, were characterized by ¹H, ¹³C, and ¹⁹F NMR spectroscopy. Kinetic studies of the catalytic reaction between <b>4a</b> and 4-octyne (<b>5b</b>) using <b>3c</b> and [Ph₃C]­[B­(C₆F₅)₄] in C₆D₅Br revealed that the catalytic reaction was zero-order for both <b>4a</b> and <b>5b</b>, indicating that the rate-determining step involved the C­(sp³)–H bond activation of <b>4a</b> by vinylhafnium intermediate <b>11c</b>

Requirement of NEU3 sialidase activity for enhanced phosphorylation of EGFR and Src.

<p>(A) The sialidase activities of NEU3 wild type and the mutant (Y370 or N88D) in the EGFR-overexpressing clones were assayed with ganglioside as a substrate in independent experiments performed in triplicate (mean ±SD). (B) The phosphorylation levels of EGFR, Src and ERK1/2 in the NEU3 wild and mutant clones in EGFR-cells were measured by western blotting using the respective antibodies. Each value shown under the blot represents as a value relative to that in the vector controls.</p

NEU3-mediated potentiation of cell growth assessed by MTT assays and colony formation assays.

<p>(A) The cell growth curves of NEU3-transfected cells were compared with those of vector controls in the absence and presence of murine EGF (20 ng/ml). Three independent experiments were performed (mean ±SD). (B) The cell growth curves of EGFR- and EGFR/NEU3-transfected cells are shown with or without EGF in independent experiments performed in triplicate (mean ±SD). (C) Colony formation assays in the transfectants. The cells were plated at 1000 cells/well in six-well dishes with or without EGF, and the colonies were quantified after 7–14 days of culture. Representative images are shown. (D) Values represent means with standard deviations obtained from three independent experiments. In the right graph, the number of colonies over 3.0 mm in size was counted in independent experiments performed in triplicate (mean ±SD).</p

Two Types of Two-Component Gels Formed from Pseudoenantiomeric Ethynylhelicene Oligomers

Two-component gels formed from pseudoenantiomeric ethynylhelicene oligomers in toluene exhibited two different properties depending on difference in numbers of helicenes in the two components. The combinations (<i>M</i>)-<b>5/</b>(<i>P</i>)-<b>4</b>, (<i>M</i>)-<b>6/</b>(<i>P</i>)-<b>4</b>, and (<i>M</i>)-<b>7/</b>(<i>P</i>)-<b>4</b>, which contained oligomers with comparable numbers of helicenes, formed transparent gels (Type I gels). The combinations (<i>M</i>)-<b>6/</b>(<i>P</i>)-<b>3</b>, (<i>M</i>)-<b>7/</b>(<i>P</i>)-<b>3</b>, and (<i>M</i>)-<b>8/</b>(<i>P</i>)-<b>3</b>, which contained oligomers with considerably different numbers of helicenes, formed turbid gels (Type II gels). Negative Cotton effects were observed for the Type I gels in the region between 350 and 450 nm, and were positive for the Type II gels, despite the use of (<i>M</i>)-oligomers for the longer components. UV/vis exhibited absorption maxima at 350 nm for the Type I gels and at 338 nm for the Type II gels. Different behaviors in gel formation processes were observed by fluorescence studies. Atomic force microscopy analysis showed fiber structures of 25–50 nm diameter for Type I gels and bundles of 100–150 nm diameter for Type II gels. The stoichiometry in gel formation also differed: The Type I gels showed 1:1 stoichiometry of the two components; the Type II gels showed no 1:1 stoichiometry, likely 1:2 stoichiometry. Using the Type I and II gels, two-layer gel systems were constructed