Transition Metal-Catalyzed Tandem Electrocyclization Migration Reaction Using Styryl Azides

This thesis describes transition metal-catalyzed tandem electrocyclization reaction employing styryl azides as the nitrogen source to form highly functionalized N-heterocycles. Chapter one briefly highlights methods that construct new C–C bond or C–N bond via domino electrocyclization migration reaction. Nazarov reaction, Rantenstrauch rearrangement and transition metal catalyzed electrocyclization migration using styryl azide as nitrogen sources are discussed. In Chapter two, I show that dirhodium(II) carboxylate complex can trigger a preferential aminomethylene [1,2]-shift when exposed to β,β-dimethylene-substituted styryl azides, completing the migratorial aptitude scale. In Chapter three and four, I describe my effort to explore the migration selectivity of ester and how steric environment influence the reaction outcome. I also extend the method to oxindole synthesis. Chapter five describes a way to manipulate the selectivity of sp3 C–H bond amination and electrocycliztion by controlling reaction conditions and functionality on substrates. Using certain catalyst can achieve selective transformation

Rh₂(II)-Catalyzed Ester Migration to Afford 3<i>H</i>‑Indoles from Trisubstituted Styryl Azides

Rh₂(II)-Complexes trigger the formation of 3<i>H</i>-indoles from <i>ortho</i>-alkenyl substituted aryl azides. This reaction occurs through a 4π-electron-5-atom electrocyclization of the rhodium <i>N</i>-aryl nitrene followed by a [1,2]-migration to afford only 3<i>H</i>-indoles. The selectivity of the migration is dependent on the identity of the β-styryl substituent

Rh₂(II)-Catalyzed Selective Aminomethylene Migration from Styryl Azides

Rh₂(II)-Carboxylate complexes were discovered to promote the selective migration of aminomethylenes in β,β-disubstituted styryl azides to form 2,3-disubstituted indoles. Mechanistic data are also presented that suggest that the migration occurs stepwise before diffusion of the iminium ion

Rh₂(II)-Catalyzed Selective Aminomethylene Migration from Styryl Azides

Rh₂(II)-Carboxylate complexes were discovered to promote the selective migration of aminomethylenes in β,β-disubstituted styryl azides to form 2,3-disubstituted indoles. Mechanistic data are also presented that suggest that the migration occurs stepwise before diffusion of the iminium ion

Rh₂(II)-Catalyzed Selective Aminomethylene Migration from Styryl Azides

Rh₂(II)-Carboxylate complexes were discovered to promote the selective migration of aminomethylenes in β,β-disubstituted styryl azides to form 2,3-disubstituted indoles. Mechanistic data are also presented that suggest that the migration occurs stepwise before diffusion of the iminium ion

Control of the Chemoselectivity of Metal <i>N</i>‑Aryl Nitrene Reactivity: C–H Bond Amination versus Electrocyclization

A mechanism study to identify the elements that control the chemoselectivity of metal-catalyzed N-atom transfer reactions of styryl azides is presented. Our studies show that the proclivity of the metal <i>N</i>-aryl nitrene to participate in sp³-C–H bond amination or electrocyclization reactions can be controlled by either the substrate or the catalyst. Electrocyclization is favored for mono-β-substituted and sterically noncongested styryl azides, whereas sp³-C–H bond amination through an H-atom abstraction–radical recombination mechanism is preferred when a tertiary allylic reaction center is present. Even when a weakened allylic C–H bond is present, our data suggest that the indole is still formed through an electrocyclization instead of a common allyl radical intermediate. The site selectivity of metal <i>N</i>-aryl nitrenes was found to be controlled by the choice of catalyst: Ir­(I)-alkene complexes trigger electrocyclization processes while Fe­(III) porphyrin complexes catalyze sp³-C–H bond amination in substrates where Rh₂(II) carboxylate catalysts provide both products

PI3K signaling in response to Rab5 isoform depletion.

<p>A) HeLa cells were transfected with GFP (as negative control) or Rab5 isoform-specific siRNA. 48 hours post-transfection, cells were starved and then stimulated with EGF for indicated times. Cell lysates were subjected to SDS-PAGE and probed with antibodies as indicated. Band intensity was quantified with AlphaEaseFc 4.0 software. Bars represent the mean value ± S.E. from four independent experiments. Analysis was carried out with a two-way ANOVA, Bonferroni’s post-test. P<0.05. B) HeLa cells were transfected with indicated siRNA. 48 hours post-transfection, cells were seeded onto micropatterned coverslips coated with fibronectin, and then allowed to spread out for 2 hours in starvation medium. Starved cells were stimulated with 10 % FCS for 3 minutes and then fixed for PIP₃-FITC antibody immuno-staining. Images shown here are average projections of PIP₃ staining from 30–35 cells.</p

Rab5C depletion significantly inhibits cell migration.

<p>A) DIC images of stable Rab5 isoform knock-down (KD) HeLa cells taken with light microscope at 40X magnification (left panel). Arrows indicate membrane ruffles. KD of Rab5 isoforms (right panel) in these stable cell lines is shown in the immunoblots following SDS-PAGE as described in Experimental Procedures. B) 0.5–1 mm width wounds were made on a monolayer of HeLa stable control or Rab5 isoform KD cells. 5–7 wounded spots in each dish were imaged with time-lapse microscope every 5 minutes for 20 hours. C) The percentage of wound closure (left panel) was calculated from images acquired at time 0 and 16 hours with ImageJ. For each sample, at least 5 images were used to calculate the percentage of wound closure in each experiment. The graph represents the Mean± S.E. from four independent experiments. U937 cells (right panel) transiently transfected with siRNA against Rab5 isoforms were seeded in the upper chamber of the Transwell plates and allowed to migrate towards 10% FBS in the bottom chamber for 24 hours. Migrated cells were measured as indicated in Material and Methods. Data are normalized to initial seeding cell numbers. The graph represents the Mean± S.E. from four independent experiments. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test.(*P<0.05, ***P<0.001)</p

Depletion of Rab5C reduces cell adhesion.

<p>A) HeLa cells were seeded on coverslips O/N and then transfected with GFP or Rab5 isoform siRNAs. The focal adhesion complex was visualized by immunostaining with vinculin antibody. The numbers of focal adhesion complexes were determined with ImageJ. The graph represents Mean± S.E. from 30 cells. Analysis was carried out with a one-way ANOVA, Dunnett’s post-test. P<0.0001. B) HeLa cells transfected with GFP or Rab5 isoform siRNAs were re-suspended and re-plated on fibronectin-coated plates for indicated times. At the end of each time point, cell lysates were extracted and prepared for SDS-PAGE and Western bloting. The activation of focal adhesion kinase was determined with phospho-FAK antibody. Total levels of FAK were not determined. The data represents Mean± S.E. from three independent experiments. Analysis was carried out with a two-way ANOVA, Bonferroni’s post-test. P<0.05.</p

Rab5 Isoforms Orchestrate a “Division of Labor” in the Endocytic Network; Rab5C Modulates Rac-Mediated Cell Motility

<div><p>Rab5, the prototypical Rab GTPase and master regulator of the endocytic pathway, is encoded as three differentially expressed isoforms, Rab5A, Rab5B and Rab5C. Here, we examined the differential effects of Rab5 isoform silencing on cell motility and report that Rab5C, but neither Rab5A nor Rab5B, is selectively associated with the growth factor-activation of Rac1 and with enhanced cell motility. Initial observations revealed that silencing of Rab5C expression, but neither Rab5A nor Rab5C, led to spindle-shaped cells that displayed reduced formation of membrane ruffles. When subjected to a scratch wound assay, cells depleted of Rab5C, but not Rab5A or Rab5B, demonstrated reduced cell migration. U937 cells depleted of Rab5C also displayed reduced cell motility in a Transwell plate migration assay. To examine activation of Rac, HeLa cells stably expressing GFP-Rac1 were independently depleted of Rab5A, Rab5B or Rab5C and seeded onto coverslips imprinted with a crossbow pattern. 3-D GFP-Rac1 images of micro-patterned cells show that GFP-Rac1 was less localized to the cell periphery in the absence of Rab5C. To confirm the connection between Rab5C and Rac activation, HeLa cells depleted of Rab5 isoforms were starved and then stimulated with EGF. Rac1 pull-down assays revealed that EGF-stimulated Rac1 activity was significantly suppressed in Rab5C-suppressed cells. To determine whether events upstream of Rac activation were affected by Rab5C, we observed that EGF-stimulated Akt phosphorylation was suppressed in cells depleted of Rab5C. Finally, since spatio-temporal assembly/disassembly of adhesion complexes are essential components of cell migration, we examined the effect of Rab5 isoform depletion on the formation of focal adhesion complexes. Rab5C-depleted HeLa cells have significantly fewer focal adhesion foci, in accordance with the lack of persistent lamellipodial protrusions and reduced directional migration. We conclude that Rab5 isoforms selectively oversee the multiple signaling and trafficking events associated with the endocytic network.</p></div