Noninnocence of Indigo: Dehydroindigo Anions as Bridging Electron-Donor Ligands in Diruthenium Complexes

Complexes of singly or doubly deprotonated indigo (H₂Ind) with one or two [Ru­(pap)₂]²⁺ fragments (pap = 2-phenylazopyridine) have been characterized experimentally [molecular structure, voltammetry, electron paramagnetic resonance (EPR), and UV–vis–near-IR spectroelectrochemistry] and by time-dependent density functional theory calculations. The compound [Ru­(pap)₂(HInd^–)]­ClO₄ ([<b>1</b>]­ClO₄) was found to contain an intramolecular NH---O hydrogen bond, whereas [{Ru­(pap)₂}₂(μ-Ind^2–)]­(ClO₄)₂ ([<b>2</b>]­(ClO₄)₂), isolated as the meso diastereoisomer with near-IR absorptions at 1162 and 991 nm, contains two metals bridged at 6.354 Å distance by the bischelating indigo dianion. The spectroelectrochemical study of multiple reversible reduction and oxidation processes of <b>2</b>^<i>n</i> (<i>n</i> = 4+, 3+, 2+, 1+, 0, 1–, 2–, 3–, 4−) reveals the stepwise addition of electrons to the terminal π-accepting pap ligands, whereas the oxidations occur predominantly at the anionic indigo ligand, producing an EPR-identified indigo radical intermediate and revealing the suitability of deprotonated indigo as a σ- and π-donating bischelating bridge

Metal–Metal Bridging Using the DPPP Dye System: Electronic Configurations within Multiple Redox Series

Redox series [L_<i>n</i>Ru­(μ-DPPP)­RuL_<i>n</i>]^<i>k</i>, H₂DPPP = 2,5-dihydro-3,6-di-2-pyridylpyrrolo­(3,4-<i>c</i>)­pyrrole-1,4-dione and L = 2,4-pentanedionato (acac^–), 2,2′-bipyridine (bpy), and 2-phenylazopyridine (pap), have been studied by voltammetry (CV, DPV), EPR, and UV–vis–NIR spectroelectrochemistry, supported by TD-DFT calculations. Crystal structure analysis and ¹H NMR revealed oxidation states [(acac)₂Ru^III(μ-DPPP^2–)­Ru^III(acac)₂] and [(bpy)₂Ru^II(μ-DPPP^2–)­Ru^II(bpy)₂]²⁺ for the corresponding precursors, isolated as <i>rac</i> diastereomers. Oxidation was observed to occur mainly at the bridging ligand (DPPP^2– → DPPP^•–), whereas the site of reduction (DPPP, Ru, or L) depends on effects from the ancillary ligands L. The metal coordination of a derivative of the pigment forming 2,5-dihydro-pyrrolo­(3,4-<i>c</i>)­pyrrole-1,4-dione (DPP) dyes and the analysis of corresponding multistep redox series add to the previously recognized coordinative and electron transfer potential of dye molecules of the azo, indigo, anthraquinone, and formazanate type

Isomeric Diruthenium Complexes of a Heterocyclic and Quinonoid Bridging Ligand: Valence and Spin Alternatives for the Metal/Ligand/Metal Arrangement

5,7,12,14-Tetraazapentacene-6,13-quinone (L) reacts with 2 equiv of [Ru­(acac)₂(CH₃CN)₂] to form two linkage isomeric bis­(chelate) compounds, [{Ru^II(acac)₂}₂(μ-L)], blue <b>1</b>, with 5,6;12,13 coordination and violet <b>2</b> with 5,6;13,14 coordination. The linkage isomers could be separated, structurally characterized in crystals as <i>rac</i> diastereomers (ΔΔ/ΛΛ), and studied by voltammetry (CV, DPV), EPR, and UV–vis–NIR spectroelectrochemistry (<i>meso</i>-<b>1</b>, <i>rac</i>-<b>2</b>). DFT and TD-DFT calculations support the structural and spectroscopic results and suggest a slight energy preference (Δ<i>E</i> = 263 cm^–1) for the <i>rac</i>-isomer <b>1</b> as compared to <b>2</b>. Starting from the Ru^II–(μ-L⁰)–Ru^II configurations of <b>1</b> and <b>2</b> with low-lying metal-to-ligand charge transfer (MLCT) absorptions, the compounds undergo two reversible one-electron oxidation steps with open-shell intermediates <b>1</b>^<b>+</b> (<i>K</i>_c = 4 × 10⁴) and <b>2</b>^<b>+</b> (<i>K</i>_c = 6 × 10⁵). Both monocations display metal-centered spin according to EPR, but the DFT-calculated spin densities suggest a Ru^III(μ-L^•–)­Ru^III three-spin situation with opposite spin density at the bridging ligand for the <i>meso</i> form of <b>1</b>⁺, estimated to lie 1887 cm^–1 lower in energy than <i>rac</i>-<b>1</b>^<b>+</b>, which is calculated with a Class II mixed-valent situation Ru^III–(μ-L⁰)–Ru^II. A three-spin arrangement Ru^III–(μ-L^•–)–Ru^III with negative spin density at one metal site is suggested by DFT for <i>rac</i>-<b>2</b>^<b>+</b> which is more stable by Δ<i>E</i> = 890 cm^–1 than <i>rac</i>-<b>1</b>^<b>+</b>. Reduction of <b>1</b> or <b>2</b> (<i>K</i>_c = 10⁷–10⁸) occurs mainly at the central bridging ligand with notable contributions (30%) from the metals in <b>1</b>^<b>–</b> and <b>2</b>^<b>–</b>. The mixed-valent Ru^III(μ-L)­Ru^II versus radical-bridged Ru^III(μ-L^•–)­Ru^III alternative is discussed comprehensively in comparison with related valence-ambiguous cases

1,5-Diamido-9,10-anthraquinone, a Centrosymmetric Redox-Active Bridge with Two Coupled β‑Ketiminato Chelate Functions: Symmetric and Asymmetric Diruthenium Complexes

The dinuclear complexes {(μ-H₂L)­[Ru­(bpy)₂]₂}­(ClO₄)₂ ([<b>3</b>]­(ClO₄)₂), {(μ-H₂L)­[Ru­(pap)₂]₂}­(ClO₄)₂ ([<b>4</b>]­(ClO₄)₂), and the asymmetric [(bpy)₂Ru­(μ-H₂L)­Ru­(pap)₂]­(ClO₄)₂ ([<b>5</b>]­(ClO₄)₂) were synthesized via the mononuclear species [Ru­(H₃L)­(bpy)₂]­ClO₄ ([<b>1</b>]­ClO₄) and [Ru­(H₃L)­(pap)₂]­ClO₄ ([<b>2</b>]­ClO₄), where H₄L is the centrosymmetric 1,5-diamino-9,10-anthraquinone, bpy is 2,2′-bipyridine, and pap is 2-phenylazopyridine. Electrochemistry of the structurally characterized [<b>1</b>]­ClO₄, [<b>2</b>]­ClO₄, [<b>3</b>]­(ClO₄)₂, [<b>4</b>]­(ClO₄)₂, and [<b>5</b>]­(ClO₄)₂ reveals multistep oxidation and reduction processes, which were analyzed by electron paramagnetic resonance (EPR) of paramagnetic intermediates and by UV–vis–NIR spectro-electrochemistry. With support by time-dependent density functional theory (DFT) calculations the redox processes could be assigned. Significant results include the dimetal/bridging ligand mixed spin distribution in <b>3</b>³⁺ versus largely bridge-centered spin in <b>4</b>³⁺a result of the presence of Ru^II-stabilizig pap coligands. In addition to the metal/ligand alternative for electron transfer and spin location, the dinuclear systems allow for the observation of ligand/ligand and metal/metal site differentiation within the multistep redox series. DFT-supported EPR and NIR absorption spectroscopy of the latter case revealed class II mixed-valence behavior of the oxidized asymmetric system <b>5</b>³⁺ with about equal contributions from a radical bridge formulation. In comparison to the analogues with the deprotonated 1,4-diaminoanthraquinone isomer the centrosymmetric H₂L^2– bridge shows anodically shifted redox potentials and weaker electronic coupling between the chelate sites

1,5-Diamido-9,10-anthraquinone, a Centrosymmetric Redox-Active Bridge with Two Coupled β‑Ketiminato Chelate Functions: Symmetric and Asymmetric Diruthenium Complexes

The dinuclear complexes {(μ-H₂L)­[Ru­(bpy)₂]₂}­(ClO₄)₂ ([<b>3</b>]­(ClO₄)₂), {(μ-H₂L)­[Ru­(pap)₂]₂}­(ClO₄)₂ ([<b>4</b>]­(ClO₄)₂), and the asymmetric [(bpy)₂Ru­(μ-H₂L)­Ru­(pap)₂]­(ClO₄)₂ ([<b>5</b>]­(ClO₄)₂) were synthesized via the mononuclear species [Ru­(H₃L)­(bpy)₂]­ClO₄ ([<b>1</b>]­ClO₄) and [Ru­(H₃L)­(pap)₂]­ClO₄ ([<b>2</b>]­ClO₄), where H₄L is the centrosymmetric 1,5-diamino-9,10-anthraquinone, bpy is 2,2′-bipyridine, and pap is 2-phenylazopyridine. Electrochemistry of the structurally characterized [<b>1</b>]­ClO₄, [<b>2</b>]­ClO₄, [<b>3</b>]­(ClO₄)₂, [<b>4</b>]­(ClO₄)₂, and [<b>5</b>]­(ClO₄)₂ reveals multistep oxidation and reduction processes, which were analyzed by electron paramagnetic resonance (EPR) of paramagnetic intermediates and by UV–vis–NIR spectro-electrochemistry. With support by time-dependent density functional theory (DFT) calculations the redox processes could be assigned. Significant results include the dimetal/bridging ligand mixed spin distribution in <b>3</b>³⁺ versus largely bridge-centered spin in <b>4</b>³⁺a result of the presence of Ru^II-stabilizig pap coligands. In addition to the metal/ligand alternative for electron transfer and spin location, the dinuclear systems allow for the observation of ligand/ligand and metal/metal site differentiation within the multistep redox series. DFT-supported EPR and NIR absorption spectroscopy of the latter case revealed class II mixed-valence behavior of the oxidized asymmetric system <b>5</b>³⁺ with about equal contributions from a radical bridge formulation. In comparison to the analogues with the deprotonated 1,4-diaminoanthraquinone isomer the centrosymmetric H₂L^2– bridge shows anodically shifted redox potentials and weaker electronic coupling between the chelate sites

1,5-Diamido-9,10-anthraquinone, a Centrosymmetric Redox-Active Bridge with Two Coupled β‑Ketiminato Chelate Functions: Symmetric and Asymmetric Diruthenium Complexes

The dinuclear complexes {(μ-H₂L)­[Ru­(bpy)₂]₂}­(ClO₄)₂ ([<b>3</b>]­(ClO₄)₂), {(μ-H₂L)­[Ru­(pap)₂]₂}­(ClO₄)₂ ([<b>4</b>]­(ClO₄)₂), and the asymmetric [(bpy)₂Ru­(μ-H₂L)­Ru­(pap)₂]­(ClO₄)₂ ([<b>5</b>]­(ClO₄)₂) were synthesized via the mononuclear species [Ru­(H₃L)­(bpy)₂]­ClO₄ ([<b>1</b>]­ClO₄) and [Ru­(H₃L)­(pap)₂]­ClO₄ ([<b>2</b>]­ClO₄), where H₄L is the centrosymmetric 1,5-diamino-9,10-anthraquinone, bpy is 2,2′-bipyridine, and pap is 2-phenylazopyridine. Electrochemistry of the structurally characterized [<b>1</b>]­ClO₄, [<b>2</b>]­ClO₄, [<b>3</b>]­(ClO₄)₂, [<b>4</b>]­(ClO₄)₂, and [<b>5</b>]­(ClO₄)₂ reveals multistep oxidation and reduction processes, which were analyzed by electron paramagnetic resonance (EPR) of paramagnetic intermediates and by UV–vis–NIR spectro-electrochemistry. With support by time-dependent density functional theory (DFT) calculations the redox processes could be assigned. Significant results include the dimetal/bridging ligand mixed spin distribution in <b>3</b>³⁺ versus largely bridge-centered spin in <b>4</b>³⁺a result of the presence of Ru^II-stabilizig pap coligands. In addition to the metal/ligand alternative for electron transfer and spin location, the dinuclear systems allow for the observation of ligand/ligand and metal/metal site differentiation within the multistep redox series. DFT-supported EPR and NIR absorption spectroscopy of the latter case revealed class II mixed-valence behavior of the oxidized asymmetric system <b>5</b>³⁺ with about equal contributions from a radical bridge formulation. In comparison to the analogues with the deprotonated 1,4-diaminoanthraquinone isomer the centrosymmetric H₂L^2– bridge shows anodically shifted redox potentials and weaker electronic coupling between the chelate sites

CASP led to increased expression of DR5 and TRAIL.

<p>DR5 was predominantly expressed on neutrophil cell surface. A: The fraction of neutrophils within the spleen was determined by FACS analyses in non-treated mice (no treatment) versus septic mice 20 hours after induction of CASP (CASP). Box plots are shown. n = 5/group. Results are representative of three independent experiments. B: DR5-expression as well as Ly6G-expression by murine splenocytes was determined by FACS analysis (n = 5). The expression of DR5 on the cell surface of Ly6G+ -splenocytes was compared to the expression of DR5 on all splenocytes. Boxplots are shown. Isotype controls were used for background staining. One of two experiments in which similar results were obtained is shown. C: Representative FACS density plots of the expression of DR5 by splenocytes are shown. Plots were gated on B220 and Ly6G respectively. D: TRAIL-binding on the cell surface of splenocytes is shown as determined by FACS analysis of spleens of untreated and septic mice (CASP; 20 h after CASP) (n = 5/group). Isotype controls were used for background staining. Box plots and outliers are depicted. TRAIL-expression was significantly increased during CASP. Results are representative of three independent experiments. E: TRAIL was stained in spleens of septic TRAIL-treated mice via immunohistochemistry. TRAIL was mainly detected in cells of the splenic red pulp (brown coloured cells, n = 5). One representative picture of five is depicted. A 200x magnification is shown. *p<0.05.</p

TRAIL-treatment did not influence cell viability <i>in vitro</i>.

<p>LPS-stimulation increased TRAIL-expression by splenocytes. A: Cultures of splenocytes were stimulated with TRAIL (100 ng/ml) for 48 hours. Cell viability was determined using a CellTiter Blue Assay. Box plots and outliers are depicted. TRAIL-stimulation did not alter the viability of splenocytes. n = 5/group; results are representative of two independently performed experiments. B: Cultures of splenocytes were stimulated with LPS (1 µg/ml) for 24 hours. TRAIL-expression was determined by FACS analyses. Isotype controls were used for background staining. Box plots and representative histograms of FACS analyses are shown. LPS stimulation significantly increased the expression of TRAIL on the cell surface of splenocytes. One of two experiments in which similar results were obtained is shown. *: p<0.05.</p

TRAIL-treatment improved survival of CASP.

<p>This effect was abrogated by depleting neutrophils. A: Survival of CASP is depicted as Kaplan Meier curves. Mice were treated with anti-Ly6G 24 hrs before CASP induction (anti-Ly6G) to deplete neutrophils. Controls received appropriate isotype controls (isotype). Neutrophil-depleted (anti-Ly6G, TRAIL) and untreated mice (TRAIL) received TRAIL (1 µg/g (wt/wt)) 1 h, 24 h and 48 h after CASP intravenously. TRAIL treatment significantly improved survival of sepsis in previously untreated mice (p<0.001). However, TRAIL-treatment was ineffective in Ly6G-depleted mice. B: Depletion of neutrophils was confirmed via FACS analyses. Representative data 48 hrs after neutrophil depletion are shown. The oval indicates neutrophils detected via CD11b+Ly6Cmed expression.</p

TRAIL-treatment led to induction of apoptosis in neutrophils in sepsis.

<p>A) Spleens, livers and lungs of septic saline-treated (CASP+saline) and septic TRAIL-treated (CASP+TRAIL) were analyzed 20 h after induction of CASP. Sections were stained for Ly6G. Ly6G-positive cells of respective organs (n = 5/group for each organ) were counted in three HPFs and the mean was calculated. The number of neutrophils per HPF is depicted. Box plots and outliers are shown. The infiltration of neutrophils within the septic organs is significantly decreased by TRAIL-treatment in sepsis. Results are representative of two independent experiments. B) The number of apoptotic cells within the spleen, liver and lungs was determined by immunohistochemistry (n = 5/group for each organ, mean of 3 HPFs). TUNEL-straining was performed 20 hours after CASP. Box plots and outliers are shown. TRAIL-treatment decreased the number of apoptotic cells. Results are representative of two experiments performed independently. C) Apoptotic neutrophils were detected by staining Ly6G and TUNEL. The number of apoptotic neutrophils within the respective septic organs 20 hrs after induction of CASP was counted in three HPFs and the mean was calculated (n = 5/group for each organ). Additionally, the number of total apoptotic cells per HPF was counted. The ratio of apoptotic neutrophils over all apoptotic cells was calculated for each HPF. Box plots and outliers are depicted. TRAIL-treatment increased the fraction of apoptotic neutrophils 20 hrs after induction of CASP within the septic organs. D) Representative immunohistochemical analysis of Ly6G (green) and TUNEL (red) in spleens of septic mice 20 hrs after induction of CASP with (right) and without (left) TRAIL-treatment. Apoptotic neutrophils appear yellow. *p<0.05.</p