A Catalytic Enantioselective Conjugate Addition of Cyanide to Enones

A Catalytic Enantioselective Conjugate Addition of Cyanide to Enone

Catalytic Enantioselective Construction of β-Quaternary Carbons via a Conjugate Addition of Cyanide to β,β-Disubstituted α,β-Unsaturated Carbonyl Compounds

Catalytic Enantioselective Construction of β-Quaternary Carbons via a Conjugate Addition of Cyanide to β,β-Disubstituted α,β-Unsaturated Carbonyl Compound

A Catalytic Enantioselective Conjugate Addition of Cyanide to Enones

A Catalytic Enantioselective Conjugate Addition of Cyanide to Enone

Function of p53 mutants in TLP-stimulated transcriptional activation.

<p>(<b>A</b>) Schematic representation of the structure of human p53. (<b>a</b>) Positions of TAD (transactivation domain), DBD (DNA-binding domain) and TD (tetramerization domain) are indicated with AA positions. Positions of mutation in the examined mutants are shown by vertical triangles. (<b>b</b>) AA residues of TAD1 in the TAD region. 22L and 23W have been reported to be critical for transactivation (TA), and 18T and 20S are phosphorylated (PH) amino acids (6). (<b>B</b>) Analysis of TLP-stimulated function for individual p53 mutants by an overexpression experiment. Cells were co-transfected with <i>p21</i> upstream promoter-carrying reporter plasmid and expression plasmid for p53/mutant alone or p53/mutant+TLP. Results are shown as relative luciferase activities (RLA). Ratio represents RLA of p53/mutant expression to RLA of p53/mutant+TLP expression. Some data were examined by statistical analysis. Since the control experiment (ctr) was performed with a vacant effector plasmid, ratios could not be obtained because measured faint luciferase activities are meaningless. (<b>C</b>) Analysis of TLP-stimulated function of representative p53 mutants by a knockdown experiment. TLP siRNA and scrambled (control) siRNA were used as depicted in the figure, and promoter activity was determined as described in panel B.</p

Examination of mutant TLPs on transcriptional activation and p53 binding.

<p>(<b>A</b>) Structural relationship between TBP and TLP. Amino acid numbers are indicated from N-termini. TLP covers the evolutionally conserved region of TBP. A putative p53-binding region in TBP deduced from deletion analyses <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090190#pone.0090190-Liu1" target="_blank">[44]</a> and its TLP counterpart (from 63 to 115) are depicted as a gray area. Positions of AAs of the TLP mutants used in this study (R86S, F100E, and F114E) are indicated with vertical arrowheads. (<b>B</b>) Transcription activation function of wild-type (WT) and mutant TLPs were assayed in native (<b>a</b>) and p53^−/− (<b>b</b>) cells. (<b>C</b>) Binding of TLP and p53. Wild-type and F100E TLPs were analyzed for the p53-bidnding ability by two-hybrid assay.</p

Effect of #22.23 mutation on cell growth and etoposide-induced cell death.

<p>(<b>A</b>) Five-hundred thousand p53^−/− cells in a dish were cultured for 24 hr. Cells were transfected with an expression plasmid for p53 (WT) or #22.23 (mut) together with a TLP expression plasmid. After 24 hr, 8×104 cells were replated and maintained. Cell numbers were counted every 24 hr (panels a–c). ctr: vacant plasmid. (<b>d</b>) Cell numbers at each time shown in panels a–c are displayed as ratios to the initial cell number. (<b>B</b>) Experiments were performed as described above, but replated cells were maintained in a medium containing 30 µM etoposide to examine the effect of TLP on apoptotic cell death (<b>a–c</b>). Numbers of remaining viable cells were counted. (<b>d</b>) Data are summarized as described above.</p

Effect of F100E mutation of TLP on the expression of endogenous <i>p21</i> gene and cell growth.

<p>(<b>A</b>) Wild-type (a) and p53^−/− cells (b) were transfected with expression vectors of wild-type and mutant (F100E) TLPs, and two species of <i>p21</i> transcripts were determined by RT-PCR as described in a legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090190#pone-0090190-g004" target="_blank">Fig. 4</a>. (<b>B</b>) Wild-type and mutant TLP-transfected native (<b>a</b>) and p53^−/− (<b>b</b>) cells were cultured for 24 hr. Cells (1×10⁵) were replated and cell numbers were counted every 24 hr. ctr: vacant plasmid.</p

Access to Electron-Deficient 2,2-Disubstituted Chromanes: A Highly Regioselective One-Pot Synthesis via an Inverse-Electron-Demand [4 + 2] Cycloaddition of <i>ortho</i>-Quinone Methides

We report the one-pot synthesis of 2,2-disubstituted chromanes with electron-withdrawing substituents. This reaction provides a simple yet efficient route to a wide range of electron-deficient chromanes in high yield and excellent regioselectivity. The reaction of salicylaldehyde with 1,1-disubstituted ethylenes smoothly furnishes these electron-deficient chromanes, which can be further transformed into functionalized chromanes or chromene. For example, BW683C was effectively synthesized from 5-chlorosalicylaldehyde with 4-chlorostyrene in two steps in excellent yield. The present reaction thus provides versatile access to functionalized electron-deficient chromanes and chromenes and therefore constitutes a promising tool for the synthesis of biologically and photochemically active molecules

Effect of #22.23 mutation on gene expression from endogenous <i>p21</i> promoters.

<p>(<b>A</b>) Two kinds of major <i>p21</i> transcripts produced from the human <i>p21</i> gene. Position of exons of <i>p21</i> alt-a and <i>p21</i> variant-1 transcripts and genomic DNA around the two <i>p21</i> promoters are schematically illustrated. Open and solid boxes represent non-coding and coding exons, respectively. Two primer sets indicated by thick arrows were used for RT-PCR to detect variant-1 and alt-a, respectively. (<b>B</b>) p53^−/− cells were transfected with expression vectors for wild-type and mutant (#22.23) p53, and two species of <i>p21</i> transcripts were determined by RT-PCR. Vector: vacant vector. RNAs of endogenous β-actin, p53 and TLP were also analyzed. (<b>C</b>) Assays for TLP-stimulated function of wild-type p53 and #22.23. (<b>a</b>) Experiments were performed as described in panel B. Cells were transfected with a TLP expression plasmid in addition to a p53 expression plasmid as indicated. ctr and vec: corresponding vacant vectors. (<b>b</b>) Amounts of intracellular p53 and #22.23 proteins were also detected by immunoblotting in addition to GAPDH and endogenous and exogenous TLPs. (<b>c</b>) Degree of increase in alt-a transcripts stimulated by exogenous TLP in p53-expressing cells. Ratios of band intensities of alt-a of panel (a) in vacant vector-introduced cells to that in TLP overexpressed cells were calculated for three kinds of cells.</p

TLP binds to p53 in solution.

<p>(<b>A</b>) Detection of p53-biding ability of TLP. TLP and TBP were examined for p53 binding by a GST pull-down assay, and affinities of both proteins against p53 were roughly determined by a competitive pull-down assay. (<b>a</b>) FH-p53 was challenged to GTS-tagged TBP (lane 1) or TLP (lane 3) as indicated and a simple pull-down assay was performed. TBP/TLP and FH-p53 were detected by α-GST antibody and α-53 antibody, respectively. No signal was detected when only GST tag was used (data not shown). FH-TLP (lane 2) and FH-TBP (lane 4) were co-applied to the GST-fused protein-adsorbed beads together with FH-p53, respectively, as competitors for GST proteins. (<b>b</b>) Relative band intensities of lane 2 (TBP+TLP), lane 3 (TLP) and lane 4 (TLP+TBP) to that of lane 1 (TBP) of panel (a) are displayed. (<b>B</b>) Comparison of p53-binding affinities of TLP and TBP. GST pull-down assays of lane 1 and lane 2 of panel A-a were performed with increasing amounts of GST-TBP (a) and GST-TLP (b), respectively. input: input protein corresponding to experimental (pull-down) materials. (<b>c</b>) Relative band intensity for p53 protein of panel (a). Results of 0.05 and 0.1 pmole of GST proteins of panel (a) are shown again in the magnified graph.</p