Diruthenium Naphthalene and Anthracene Complexes Containing a Doubly Linked Dicyclopentadienyl Ligand

The reaction of <i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(CO)₄Br₂ with naphthalene affords the <i>syn</i>-facial [<i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(μ-η⁶,η⁶-C₁₀H₈)]­[OTf]₂, (<b>2</b>^<b>2+</b>), a complex that appears to be two electrons short of the 18-electron rule. Density functional theory (DFT) calculations suggest that the Ru atoms satisfy their missing valence by a combination of a weak metal–metal bond and sharing electrons from the central π bond of the naphthalene. The one-electron reduction of <b>2</b>^<b>2+</b> yields <b>2</b>^<b>+</b>, a Class II mixed-valence complex, while the two-electron reduction of <b>2</b>^<b>2+</b> causes a hapticity change from η⁶ to η⁴ on one of the naphthalene rings and yields <i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(μ-η⁶,η⁴-C₁₀H₈<b>)</b> (<b>2</b>^<b>0</b>), a zwitterionic complex. The DFT calculations predict that the <i>C</i>_<i>s</i> isomer of <b>2⁰</b> is 4.69 kcal/mol lower in energy than the <i>C</i>_2<i>v</i> isomer, which is a transition state. Reaction of <i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(CO)₄Br₂ with anthracene affords the analogous <i>syn</i>-facial anthracene complex, [<i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(μ-η⁶,η⁶-C₁₄H₁₀)]­[OTf]₂, (<b>4</b>), and the tetranuclear dianthracene complex, [<i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(μ-η⁶,η⁶-C₁₄H₁₀)]₂[OTf]₄, (<b>5</b>). <b>2</b>^<b>2+</b>, <b>2</b>^<b>0</b>, and <b>5</b> were structurally characterized by X-ray diffraction

Diruthenium Naphthalene and Anthracene Complexes Containing a Doubly Linked Dicyclopentadienyl Ligand

The reaction of <i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(CO)₄Br₂ with naphthalene affords the <i>syn</i>-facial [<i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(μ-η⁶,η⁶-C₁₀H₈)]­[OTf]₂, (<b>2</b>^<b>2+</b>), a complex that appears to be two electrons short of the 18-electron rule. Density functional theory (DFT) calculations suggest that the Ru atoms satisfy their missing valence by a combination of a weak metal–metal bond and sharing electrons from the central π bond of the naphthalene. The one-electron reduction of <b>2</b>^<b>2+</b> yields <b>2</b>^<b>+</b>, a Class II mixed-valence complex, while the two-electron reduction of <b>2</b>^<b>2+</b> causes a hapticity change from η⁶ to η⁴ on one of the naphthalene rings and yields <i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(μ-η⁶,η⁴-C₁₀H₈<b>)</b> (<b>2</b>^<b>0</b>), a zwitterionic complex. The DFT calculations predict that the <i>C</i>_<i>s</i> isomer of <b>2⁰</b> is 4.69 kcal/mol lower in energy than the <i>C</i>_2<i>v</i> isomer, which is a transition state. Reaction of <i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(CO)₄Br₂ with anthracene affords the analogous <i>syn</i>-facial anthracene complex, [<i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(μ-η⁶,η⁶-C₁₄H₁₀)]­[OTf]₂, (<b>4</b>), and the tetranuclear dianthracene complex, [<i>cis</i>-{(η⁵-C₅H₃)₂(CMe₂)₂}­Ru₂(μ-η⁶,η⁶-C₁₄H₁₀)]₂[OTf]₄, (<b>5</b>). <b>2</b>^<b>2+</b>, <b>2</b>^<b>0</b>, and <b>5</b> were structurally characterized by X-ray diffraction

Differential Expression of Neural Progenitor, Müller glia Regeneration, and Glioma Genes in <i>Tg(flk1:RFP)is18/+</i> tumor progression.

<p>*Raymond et al., 2006.</p>#<p>Kassen el al., 2009; Nelson et al., 2012; Ramachandran et al., 2011; Ramachandran et al., 2012; Wan et al., 2012.</p>$<p>Zhao et al., 2010; Lin et al., 2012.</p><p>Differential Expression of Neural Progenitor, Müller glia Regeneration, and Glioma Genes in <i>Tg(flk1:RFP)is18/+</i> tumor progression.</p

Characterization of <i>lincRNAis18</i> tumors indicates a glial cell origin.

<p>Immunolabeling and in situ hybridization of cryosections from wild type retina (A–D, I, J, M–P) and advanced <i>lincRNAis18</i> tumors (E–H, K, L, Q–T). Cells in rosettes in the tumors label with neurofilament marker RT-97 (red) and photoreceptor marker recoverin (green) (E, F). The synaptic vesicle marker SV2 (green), which is enriched in the retinal plexiform layers (C), was distributed throughout the fibrous tumor mass (G). (D) BrdU (green) incorporated into proliferating progenitor cells at the ciliary marginal zone of the wild type retina (arrow). (H) Intense labeling of BrdU incorporation was detected in cells forming rosettes and throughout the tumor mass. In wild type the glial marker GFAP is most evident in the Müller glia end feet that sit in the retinal ganglion cell layer (I–L). GFAP expression is absent in the oligodendrocytes and astrocytes of the optic nerve (I, K arrow). In contrast, GFAP was readily detected in long streaks throughout the tumor tissue (M, N, P), and was expressed by cells in the mutant optic nerve (O, arrow). BLBP is present at the ciliary marginal zone of wild type retina (L) and appeared present throughout the tumor mass (P). A, E, I, M, Differential interference contrast (DIC) overlay on immunofluorescence labeling images. CMZ, ciliary marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; ON, optic nerve; ONH, optic nerve head; ONL, outer nuclear layer. All scale bars represent 50 µm, except in panels K and O scale bars represent 100 µm.</p

Histological analysis of <i>Tg(flk1:RFP)is18</i> tumors reveals similarities with retinoblastoma and glioma.

<p>Coronal sections through heads of wild type (A, E–H) and <i>Tg(flk1:RFP)is18/+</i> adults (B–D, I–L). (B) 5-month-old <i>Tg(flk1:RFP)is18/+</i> adult with retinal tumor filling the vitreous. 1-year-old <i>Tg(flk1:RFP)is18/+</i> adults with unilateral (C) or bilateral tumors (D). Tumor cells extend through the optic pathway to the tectum. (E,F) Ciliary marginal zone and mature retina in wild type. (G) Fibrillar ribbon-like structure of a wild type optic nerve exiting the eye. (H) Section through forebrain shows the sacus dorsalis and left and right lobes of the anterior region of the optic tectum. (I) Intact ciliary marginal zone in the tumorous retina from <i>Tg(flk1:RFP)is18/+</i> adult shown in B. Degeneration of the retinal pigment epithelium and photoreceptor outer segments is evident. Disorganization of the retinal layers adjacent to the ciliary marginal zone is present. Streaking across the inner plexiform layer appears similar to reactive Müller glia. (J) Advanced tumors contain rosettes and blood vessels extending throughout the tumor tissue. (K) Disorganization and dysplasia in the optic nerve of the <i>Tg(flk1:RFP)is18/+</i> adult in C. (L) Brain from the adult in panel D showing dysplasia of the optic tract with infiltration and disruption of normal brain structures. (M) Dysplastic areas of advanced tumor with rosettes of various sizes (arrowheads) and extensive glial fibrillar proliferation (arrow). (N) “Salt and pepper” chromatin dispersion pattern (arrowheads) consistent with a neuroectodermal tumor cell. (O) Disorganization of the optic nerve with absence of organized fibrils. (P) Expansion of the optic lobe with possible areas of necrosis (arrowheads). CMZ, ciliary marginal zone; GCL, ganglion cell layer; INL, inner nuclear layer; OL, optic lobe; ON, optic nerve; ONL, outer nuclear layer; OT, optic tract. Scale bars A, B, C, D 500 µm; E, F, G, I, J, N 20 µm; H, K, L 100 µm; M, O, P 50 µm.</p

Linkage of <i>Tg(flk1:RFP)is18</i> transgene integration site to position 24.219 Mb Chromosome 3.

<p>*Microsatellite STS markers Z7419, G39247, Z5197, Z7486. <i>cbx1a STS</i> is a Short Simple Repeat located in the 5th intron of the <i>cbx1a</i> gene. Genotype determined by size of PCR amplification products after gel electrophoresis. L, long; M, middle; S, short allele PCR products. <i>is18</i> transgenic genotype (+ or −) confirmed by PCR with primers that amplify across the genomic DNA-transgene junction (3) or amplify an internal fragment of the transgene concatemer (4).</p>#<p>Genotype of <i>is18</i>/+ female and WIK male was determined. Segregation of STS markers in 10 is18/+ and 10+/+ siblings was used to determine the haplotype of the chromosome containing the is18 transgene integration. The <i>Tg(RFP)is18</i> chromosome haplotype in the region surrounding the integration site is Z7419-L, G39247-S, Z5197-L, <i>cbx1a</i> STS-L.</p>$<p>100 <i>is18</i>/+ and 100+/+ siblings from a cross between a <i>Tg(flk1:RFP)is18/+</i> female and WIK wild type male were genotyped by PCR. 1/100 <i>Tg(flk1:RFP)is18/+</i> progeny was homozygous Z7419 M/M and heterozygous G39247 L/S. This indicates a single recombination event between markers Z7419 and G39247 and genetic map distance of 1 centimorgan.</p><p>Linkage of <i>Tg(flk1:RFP)is18</i> transgene integration site to position 24.219 Mb Chromosome 3.</p

Categories of biological processes altered in <i>Tg(flk:RFP)is18/+</i> Pre-tumor and Tumor tissues.

<p>*DGE Group: Differential Gene Expression Group. Genes were grouped according to direction of change in expression level in Pretumor and Tumor tissue.</p><p>Categories of biological processes altered in <i>Tg(flk:RFP)is18/+</i> Pre-tumor and Tumor tissues.</p

The <i>Tg(flk1:RFP)is18</i> transgene integration site on chromosome 3 disrupts expression of <i>lincRNAis18.v2</i>.

<p>(A) Integration site of the transgene array at 24.2 Mb on chromosome 3. The integration sits downstream of the <i>HoxBa</i> cluster and upstream relative to the heterochromatin binding family member <i>cbx1a</i>. <i>lincRNAis18</i> exon I overlaps with a 5′ regulatory region of the <i>cbx1a</i> gene. Positions of upstream and downstream microsatellite markers, G39247 and Z5197, and an STS/STR in <i>cbx1a</i>, are shown. (B) RT-PCR analyses with primers in exon 1 and exon 8 of <i>lincRNAis18.v2</i> showing expression of <i>lincRNAis1.v28</i> is disrupted in 6 dpf homozygous <i>Tg(flk1:RFP)is18</i> larvae. 5 individual larvae of each genotype are shown. The genotype of each larva was confirmed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114888#pone.0114888.s006" target="_blank">Figure S6</a>). Control, expression of ribosomal protein S6 kinase b, polypeptide 1, <i>rps6kb1</i>. (C) TALENs targeting exons 2 and 5 of <i>lincRNAis18</i>. (D) Predicted structure of the <i>lincRNAis18^e2e5del</i> deletion allele. Sequence of amplicon spanning exon 2 – exon 5 junction from F1 <i>lincRNAis18^e2e5del/+</i> adult genomic DNA.</p

Differential gene expression analysis of retina RNA-Seq from wild type, pre-tumor, and retinal tumor tissues.

<p>A, B Heat map representations of differentially regulated transcripts associated with tumor initiation and progression. (A) Differential gene expression of 62 Mutation-Driver genes. Tumor suppressors are in green, oncogenes are in red. (B) Differential gene expression of genes required for photoreceptor and neural function, neural progenitor proliferation, and injury induced regeneration. Scale represents log2 fold change in gene expression. (C) Comparison of absolute fold change in gene expression levels for <i>ajap1</i>, <i>ascl1a</i>, <i>atf3</i>, <i>bysl</i>, <i>hbegfa</i>, and <i>insm1a</i> measured by RNA-Seq (upper panel) and qRT-PCR (lower panel). <i>Tg(flk1:RFP)is18/+</i> Pretumor (red bars) and <i>Tg(flk1:RFP)is18/+</i> Tumor (blue bars).</p