Differentiation between Chelate Ring Inversion and Aryl Rotation in a CF₃‑Substituted Phosphine-Sulfonate Palladium Methyl Complex

The solution conformations and dynamic properties of the CF₃-sbustituted (<i>ortho</i>-phosphino­arenesulfonate)­Pd complexes (PO-CF₃)­PdMe­(L) ([PO-CF₃]⁻ = 2-{(<i>o</i>-CF₃-Ph)₂P}-4-Me-benzenesulfonate, L = 2,6-lutidine (<b>3</b>), pyridine (<b>4</b>)) were studied by NMR spectroscopy, taking particular advantage of ³¹P–¹⁹F through-space couplings and ¹H–¹H and ¹H–¹⁹F nuclear Overhauser effects. In CD₂Cl₂ solution in the temperature range of −80 to 20 °C, <b>3</b> adopts an <i>exo</i>₂ conformation. One <i>o</i>-CF₃-Ph ring is positioned such that the CF₃ group points toward Pd (<i>exo</i>) and exhibits through-space ⁴<i>J</i>_PF coupling. The other <i>o</i>-CF₃-Ph ring is positioned such that the CF₃ group points away from Pd (<i>endo</i>) and does not exhibit through-space ⁴<i>J</i>_PF coupling, and the <i>o</i>-H lies in the deshielding region near an axial site of the Pd square plane and exhibits a low-field chemical shift (δ > 9). Complex <b>4</b> exists as a 2:1 mixture of <i>exo</i>₂ and <i>exo</i>₃ isomers in CD₂Cl₂ solution at −90 °C. In <i>exo</i>₂-<b>4</b>, one CF₃ group is <i>exo</i> and exhibits through-space ⁴<i>J</i>_PF coupling, while the other CF₃ group is <i>endo</i> and does not exhibit through-space ⁴<i>J</i>_PF coupling. In <i>exo</i>₃-<b>4</b>, both CF₃ groups are <i>exo</i> and exhibit through-space ⁴<i>J</i>_PF couplings. Complex <b>4</b> undergoes two dynamic processes: rotation of the axial <i>o</i>-CF₃-Ph ring (A_aR), which interconverts <i>exo</i>₂-<b>4</b> and <i>exo</i>₃-<b>4</b> (Δ<i>G</i>^⧧ = 9.9(5) kcal/mol), and chelate ring inversion (RI), which permutes the axial and equatorial <i>o</i>-CF₃-Ph rings (Δ<i>G</i>^⧧ = 21(1) kcal/mol)

Establishment of single-colony-derived DFCs.

<p>(A). Clone forming of DFCs. Limiting dilution was used for single clone selection. One single cell was observed after the procedure (a), several days later the cells formed a clone in which more than 50 cells were observed (d). (B). Morphological change of DFCs after several passages. (a). At passage 3, DFCs were fibroblast-like cells. (b). Cells became flat after more than 5 passages.</p

Infection efficiency of <i>piggyBac</i> Transposon system.

<p>(A). Schematic diagram of pMPH86, the <i>piggyBac</i> Transposon-mediated reversible vector for immortalization, that includes SV40 T-Ag and hygromycin B expression cassette. (B). Cell transducted by AdpBase or AdFLP for 24 hours. (a). DFCs transducted efficiently by adenoviral vectors AdpBase to establish immortalized dental follicle cells (iDFCs) (b). Efficient transduction of iDFCs by adenoviral vectors AdFLP to get deimmortalized dental follicle cells (dDFCs). (C). Hygromycin B selection. (a). Survival rate of cells infected with pMPH86 is 30%. (b). Survival rate of cells infected with <i>SSR#69</i> is 10%. (D). Expression of SV40T-Ag in cells infected with pMPH86 or <i>SSR#69</i>. (E). Integration of SV40T-Ag gene in cells infected with pMPH86 or <i>SSR#69</i>.</p

Adipogenic differentiation of DFCs, iDFCs and dDFCs.

<p>(A) DFCs, iDFCs and dDFCs can be induced to form adipocyte-like cells. Subconfluent DFCs, iDFCs and dDFCs were infected by adenovirus Ad PPARγ2 (a). or AdRFP (data unshown). 5 days after adipogenic induction, lipid droplets can be seen clearly under phase contrast microscope (b). Lipid droplets can be stain by oil red as compared to the control groups infected by AdRFP(c). (B) The expression of PPARγ2 after adipogenic differentiation of DFCs, iDFCs and dDFCs. Subconfluent cells were transducted by AdPPARγ2 or AdRFP as negative control. On day 5, western blotting was performed using anti- PPARγ2 antibody. Anti-GAPDH Western blotting ensures the same amount of samples loaded. Samples infected by AdRFP didn’t express PPARγ2. (D) Expression of adipogenic lineage-specific genes in DFCs, iDFCs and dDFCs stimulated by PPARγ2. The assays were done in three experiments. Note: *<i>p</i> < 0.05.</p

Morphology and cell proliferation of three types of DFCs.

<p>(A) Morphology of primary dental follicle cells (DFCs), immortalized dental follicle cells (iDFCs) and deimmortalized dental follicle cells (dDFCs). Primary DFCs and deimmortalized DFCs were seeded at 20% confluence and cultured for three passages (<i>P3</i>)(a and c). iDFCs were seeded at 20% confluence and cultured for 60 passages (<i>P60</i>) (b). (B) Cell proliferation assessed with CCK8 assay. (C) Cell growth curve.</p

Telomerase activity of three types of DFCs.

<p>iDFCs maintain high telomerase activity while the telomerase activity of dDFCs is similar to primary DFCs. Telomerase activity of primary DFCs and dDFCs was tested at <i>p1</i>, <i>p3</i>, <i>p5</i> and <i>p7</i>. Telomerase activity of iDFCs was tested at <i>p1</i>, <i>p5</i>, <i>p10</i>, <i>p30</i>, and <i>p60</i>. The telomerase activity of cells was measured by telomeric repeat amplification protocol.</p

The DFCs, iDFCs and dDFCs express mesenchymal stem cell surface markers.

<p>(A) iDFCs and dDFCs possess DFCs properties. iDFCs and dDFCs are both vimentin positive and ck14 negative just the same as DFCs. (B) DFCs, iDFCs and dDFCs express mesenchymal stem cell surface markers, i.e. CD73 (a) and CD105 (b), but lack the expression of CD34(c). The percentage of strol-1 positive cells is about 13%(d).</p

Osteogenic differentiation of DFCs, iDFCs and dDFCs.

<p>(A) Induction of alkaline phosphatase (ALP), the early stage of osteogenic differentiation marker and alizarin red, the late matrix mineralization marker in DFCs, iDFCs and dDFCs after osteogenic differentiation. Subconfluent DFCs, iDFCs and dDFCs were infected by adenovirus AdBMP9 (a) or AdGFP (data unshown). ALP activity of cells was stained on day 7 and measured quantitatively at days 3, 5 and 7 (c). (B) The expression of osteopontin after osteogenic Induction of DFCs, iDFCs and dDFCs. Cells were transducted by AdBMP9 (a) or AdGFP. Anti-GAPDH Western blotting ensures the same amount of samples loaded. Samples infected by AdGFP didn’t express osteopontin. (D) Expression of osteogenic lineage-specific genes in DFCs, iDFCs and dDFCs after being induced by BMP9. Cells were transducted with AdBMP9 or AdGFP as negative control. The assays were done in three experiments. Note: *<i>p</i> < 0.05.</p