Electronic Structure of Anilinopyridinate-Supported Ru₂⁵⁺ Paddlewheel Compounds

The electronic structures of the diruthenium compounds Ru₂(ap)₄Cl (<b>1</b>, ap = 2-anilinopyridinate) and Ru₂(ap)₄OTf (<b>2</b>) were investigated with UV–vis, resonance Raman, and magnetic circular dichroism (MCD) spectroscopies; SQUID magnetometry; and density functional theory (DFT) calculations. Both compounds have quartet spin ground states with large axial zero-field splitting of ∼60 cm^–1 that is characteristic of Ru₂⁵⁺ compounds having a (π*, δ*)³ electron configuration and a Ru–Ru bond order of ∼2.5. Two major visible absorption features are observed at ∼770 and 430 nm in the electronic spectra, the assignments of which have previously been ambiguous. Both bands have significant charge-transfer character with some contributions from d → d transitions. MCD spectra were measured to enable the identification of d → d transitions that are not easily observable by UV–vis spectroscopy. In this way, we are able to identify bands due to δ → δ* and δ → π* transitions at ∼16 100 and 11 200–12 300 cm^–1, respectively, the latter band being sensitive to the π-donating character of the axial ligand. The Ru–Ru stretches are coupled with pyridine rocking motions and give rise to observed resonance Raman peaks at ∼350 and 420 cm^–1, respectively

Accessing Ni(III)-Thiolate Versus Ni(II)-Thiyl Bonding in a Family of Ni–N₂S₂ Synthetic Models of NiSOD

Superoxide dismutase (SOD) catalyzes the disproportionation of superoxide (O₂^• –) into H₂O₂ and O₂(<i>g</i>) by toggling through different oxidation states of a first-row transition metal ion at its active site. Ni-containing SODs (NiSODs) are a distinct class of this family of metalloenzymes due to the unusual coordination sphere that is comprised of mixed N/S-ligands from peptide-N and cysteine-S donor atoms. A central goal of our research is to understand the factors that govern reactive oxygen species (ROS) stability of the Ni–S­(Cys) bond in NiSOD utilizing a synthetic model approach. In light of the reactivity of metal-coordinated thiolates to ROS, several hypotheses have been proffered and include the coordination of His1-Nδ to the Ni­(II) and Ni­(III) forms of NiSOD, as well as hydrogen bonding or full protonation of a coordinated S­(Cys). In this work, we present NiSOD analogues of the general formula [Ni­(N₂S)­(SR′)]⁻, providing a variable location (SR′ = aryl thiolate) in the N₂S₂ basal plane coordination sphere where we have introduced <i>o</i>-amino and/or electron-withdrawing groups to intercept an oxidized Ni species. The synthesis, structure, and properties of the NiSOD model complexes (Et₄N)­[Ni­(nmp)­(SPh-<i>o</i>-NH₂)] (<b>2</b>), (Et₄N)­[Ni­(nmp)­(SPh-<i>o</i>-NH₂-<i>p</i>-CF₃)] (<b>3</b>), (Et₄N)­[Ni­(nmp)­(SPh-<i>p</i>-NH₂)] (<b>4</b>), and (Et₄N)­[Ni­(nmp)­(SPh-<i>p</i>-CF₃)] (<b>5</b>) (nmp^2– = dianion of <i>N</i>-(2-mercaptoethyl)­picolinamide) are reported. NiSOD model complexes with amino groups positioned <i>ortho</i> to the aryl-S in SR′ (<b>2</b> and <b>3</b>) afford oxidized species (<b>2</b>^<b>ox</b> and <b>3</b>^<b>ox</b>) that are best described as a resonance hybrid between Ni­(III)-SR and Ni­(II)-^•SR based on ultraviolet–visible (UV-vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectroscopies, as well as density functional theory (DFT) calculations. The results presented here, demonstrating the high percentage of S­(3<i>p</i>) character in the highest occupied molecular orbital (HOMO) of the four-coordinate reduced form of NiSOD (NiSOD_red), suggest that the transition from NiSOD_red to the five-coordinate oxidized form of NiSOD (NiSOD_ox) may go through a four-coordinate Ni-^•S­(Cys) (NiSOD_ox-His_off) that is stabilized by coordination to Ni­(II)

Accessing Ni(III)-Thiolate Versus Ni(II)-Thiyl Bonding in a Family of Ni–N₂S₂ Synthetic Models of NiSOD

Superoxide dismutase (SOD) catalyzes the disproportionation of superoxide (O₂^• –) into H₂O₂ and O₂(<i>g</i>) by toggling through different oxidation states of a first-row transition metal ion at its active site. Ni-containing SODs (NiSODs) are a distinct class of this family of metalloenzymes due to the unusual coordination sphere that is comprised of mixed N/S-ligands from peptide-N and cysteine-S donor atoms. A central goal of our research is to understand the factors that govern reactive oxygen species (ROS) stability of the Ni–S­(Cys) bond in NiSOD utilizing a synthetic model approach. In light of the reactivity of metal-coordinated thiolates to ROS, several hypotheses have been proffered and include the coordination of His1-Nδ to the Ni­(II) and Ni­(III) forms of NiSOD, as well as hydrogen bonding or full protonation of a coordinated S­(Cys). In this work, we present NiSOD analogues of the general formula [Ni­(N₂S)­(SR′)]⁻, providing a variable location (SR′ = aryl thiolate) in the N₂S₂ basal plane coordination sphere where we have introduced <i>o</i>-amino and/or electron-withdrawing groups to intercept an oxidized Ni species. The synthesis, structure, and properties of the NiSOD model complexes (Et₄N)­[Ni­(nmp)­(SPh-<i>o</i>-NH₂)] (<b>2</b>), (Et₄N)­[Ni­(nmp)­(SPh-<i>o</i>-NH₂-<i>p</i>-CF₃)] (<b>3</b>), (Et₄N)­[Ni­(nmp)­(SPh-<i>p</i>-NH₂)] (<b>4</b>), and (Et₄N)­[Ni­(nmp)­(SPh-<i>p</i>-CF₃)] (<b>5</b>) (nmp^2– = dianion of <i>N</i>-(2-mercaptoethyl)­picolinamide) are reported. NiSOD model complexes with amino groups positioned <i>ortho</i> to the aryl-S in SR′ (<b>2</b> and <b>3</b>) afford oxidized species (<b>2</b>^<b>ox</b> and <b>3</b>^<b>ox</b>) that are best described as a resonance hybrid between Ni­(III)-SR and Ni­(II)-^•SR based on ultraviolet–visible (UV-vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectroscopies, as well as density functional theory (DFT) calculations. The results presented here, demonstrating the high percentage of S­(3<i>p</i>) character in the highest occupied molecular orbital (HOMO) of the four-coordinate reduced form of NiSOD (NiSOD_red), suggest that the transition from NiSOD_red to the five-coordinate oxidized form of NiSOD (NiSOD_ox) may go through a four-coordinate Ni-^•S­(Cys) (NiSOD_ox-His_off) that is stabilized by coordination to Ni­(II)

Accessing Ni(III)-Thiolate Versus Ni(II)-Thiyl Bonding in a Family of Ni–N₂S₂ Synthetic Models of NiSOD

Superoxide dismutase (SOD) catalyzes the disproportionation of superoxide (O₂^• –) into H₂O₂ and O₂(<i>g</i>) by toggling through different oxidation states of a first-row transition metal ion at its active site. Ni-containing SODs (NiSODs) are a distinct class of this family of metalloenzymes due to the unusual coordination sphere that is comprised of mixed N/S-ligands from peptide-N and cysteine-S donor atoms. A central goal of our research is to understand the factors that govern reactive oxygen species (ROS) stability of the Ni–S­(Cys) bond in NiSOD utilizing a synthetic model approach. In light of the reactivity of metal-coordinated thiolates to ROS, several hypotheses have been proffered and include the coordination of His1-Nδ to the Ni­(II) and Ni­(III) forms of NiSOD, as well as hydrogen bonding or full protonation of a coordinated S­(Cys). In this work, we present NiSOD analogues of the general formula [Ni­(N₂S)­(SR′)]⁻, providing a variable location (SR′ = aryl thiolate) in the N₂S₂ basal plane coordination sphere where we have introduced <i>o</i>-amino and/or electron-withdrawing groups to intercept an oxidized Ni species. The synthesis, structure, and properties of the NiSOD model complexes (Et₄N)­[Ni­(nmp)­(SPh-<i>o</i>-NH₂)] (<b>2</b>), (Et₄N)­[Ni­(nmp)­(SPh-<i>o</i>-NH₂-<i>p</i>-CF₃)] (<b>3</b>), (Et₄N)­[Ni­(nmp)­(SPh-<i>p</i>-NH₂)] (<b>4</b>), and (Et₄N)­[Ni­(nmp)­(SPh-<i>p</i>-CF₃)] (<b>5</b>) (nmp^2– = dianion of <i>N</i>-(2-mercaptoethyl)­picolinamide) are reported. NiSOD model complexes with amino groups positioned <i>ortho</i> to the aryl-S in SR′ (<b>2</b> and <b>3</b>) afford oxidized species (<b>2</b>^<b>ox</b> and <b>3</b>^<b>ox</b>) that are best described as a resonance hybrid between Ni­(III)-SR and Ni­(II)-^•SR based on ultraviolet–visible (UV-vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectroscopies, as well as density functional theory (DFT) calculations. The results presented here, demonstrating the high percentage of S­(3<i>p</i>) character in the highest occupied molecular orbital (HOMO) of the four-coordinate reduced form of NiSOD (NiSOD_red), suggest that the transition from NiSOD_red to the five-coordinate oxidized form of NiSOD (NiSOD_ox) may go through a four-coordinate Ni-^•S­(Cys) (NiSOD_ox-His_off) that is stabilized by coordination to Ni­(II)

TLR3 stimulation increases GPR15 on the surface of CD4⁺ T cells.

<p>PBMCs were incubated with different ligands for TLRs and GPR15 expression on CD4⁺ T cells was analyzed. Cells were gated for lymphocytes, CD3⁺, CD4⁺ as already shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g001" target="_blank">Figure 1A</a>. The bar graph is a summary of four donors which were analysed in two independent experiments (A). Upon TLR3 triggering, GPR15 is mostly up-regulated on central memory T cells (B). To exclude cell-cell interaction effects T cells were further separated using negative selection with magnetic beads and stimulated with TLR3 ligand polyIC (C). Pre-treatment of T cells with TLR3 signalling inhibitor PepinhTRIF abrogates the increase of GPR15 on the T cell surface (D). To test if TLR3 stimulation can up-regulate other co-receptors it was also stained for CXCR6 (E), CCR5 (F) and CXCR4 (G). GPR15 expression is shown as a percent of the gated CD4⁺ T cell subpopulations. Statistical analysis was done with GraphPad Prism using paired t-test.</p

TLR3 stimulation up-regulates GPR15 also on CD8⁺ T cells and CD19⁺ B cells.

<p>The GPR15 expression was studied on whole PBMCs (A,B) or separated T and B cells (C) using additional anti-CD8 (A) or CD19 (B) antibodies. GPR15 is shown as a percent of CD8⁺ T or CD19⁺ B cells.</p

GPR15 is strongly up-regulated on gut homing CD4⁺Tcells and is highly expressed on colon CD4⁺Tcells.

<p>TLR3 stimulation up-regulates GPR15 on gut homing (α4β7-integrin⁺) (A, C) and on CD4⁺ T cells homing to lymph nodes (CD62L⁺) (B, D). The different symbols in the Figures C and D specify different donors. Before TLR3 stimulation both subsets express GPR15 to a similar level (E). The different symbols describe individual donors (E). PBMCs were isolated from whole blood by Lymphoprep gradient centrifugation and co-stained for CD4, GPR15 and CD62L or β7-integrin. The cells were gated on lymphocytes, CD4⁺ as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g001" target="_blank">Figure 1 A</a>. The graphs show GPR15 expression as a percent of CD62L⁺ CD4⁺ T cells or β7⁺ CD4⁺ T cells expressing the co-receptor. The experiments were done at least two times including two donors each time. Statistical analysis was done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g003" target="_blank">Figure 3</a> using paired t-test. Human colon intraepithelial mononuclear cells (IEMC) and lamina propria mononuclear cells (LPMC) express GPR15 on high level. IEMC and LPMC were isolated following the described protocol and co-stained for CD45, CD3, CD4 and GPR15. Cells were gated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088195#pone-0088195-g001" target="_blank">Figure 1A</a> with the accretion that CD45 were gated out to exclude epithelial cell contamination (F). Colon biopsies of HIV-1 infected and uninfected individuals were immunofluorescently stained for GPR15, CD4 and cell nuclei using DAPI. Slides were analysed by confocal microscopy. Three biopsies per patient and 15–20 images per biopsy were acquired at 63×. Cells were enumerated using ImageJ cell counting software for % of CD4⁺ cell expressing GPR15 (G).</p

GPR15 is mostly present on central memory CD4⁺Tcells in HIV-1 infected individuals and uninfected controls.

<p>PBMCs were isolated from whole blood by Lymphoprep gradient centrifugation and stained for CD3, CD4, CCR7, CD45RA and GPR15. (A) Cells were gated for lymphocytes, CD3⁺, CD4⁺ and CCR7⁺CD45RA⁻ (CM: central memory), CCR7⁻CD45RA⁻ (EM: effector memory) or CCR7⁺CD45RA⁺ (N: naïve) (A) and GPR15 expression on the subsets was analyzed via FACS (B). The GPR15 expression on CD4⁺ T cell subpopulations was analyzed in eight uninfected controls and eleven HIV-1 infected patients (C) as indicated in (A, B). GPR15 expression is shown as the percent of the analysed subpopulation which expresses the co-receptor (C). Blood samples taken two month later from the two high GPR15 expressing HIV-1 infected patients and two controls (shown in C) were stained for GPR15, CD4 and CD8 (D) or CD4 and other co-receptors like CXCR4, CCR5 and CXCR6 (E). The co-receptor expressions are shown as a percent of CD4⁺ and CD8⁺ T cells expressing GPR15 (D) or of CD4⁺ T cells expressing CXCR4, CCR5 or CXCR6 (E). Statistical analysis was done using Wilcoxon signed-rank test with GraphPad Prism.</p

HIV-1 infection increases GPR15 expression on infected cells.

<p>The PM1 T cell line was infected with three different primary HIV-1 isolates the multitropic isolates 25 and 4052 and the R5-tropic 2195 for 3 days and afterwards stained for GPR15 surface expression and intracellular p24. The percent of uninfected (p24−) or infected (p24+)cells expressing GPR15 is shown.</p

Comparison of parameters between time points, pre, immediately post, +5, +10, +15 and +20 minutes post WBC exposures for all exposures.

*<p><b>Time points for all lengths of WBC exposure: 0 = pre WBC exposure, 1 = Immediately post WBC exposure, 2 = 5 minutes post WBC exposure, 3 = 10 minutes post WBC exposure, 4 = 15 minutes WBC exposure and 5 = 20 post WBC exposure.</b></p>**<p><b>Core temperature (°C) average mean difference.</b></p>***<p><b>Tsk (°C) average mean difference.</b></p