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The size-dependent recognition of large anions by shape-persistent macrocycles is poorly understood. To address this shortcoming, we created a bulky cyanostar that only forms 1 : 1 complexes and show the affinities best follow the expectations of size matching but only when solvent bias is removed.

Here we developed a new family of Zn-containing magnetic oxides of different structures by thermal decomposition of Zn(acac)2 in the reaction solution of preformed magnetite nanoparticles (NPs) stabilized by polyphenylquinoxaline. Upon an increase of the Zn(acac)2 loading from 0.15 to 0.40 mmol (vs 1 mmol of Fe(acac)3), the Zn content increases, and the Zn-containing magnetic oxide NPs preserve a spinel structure of magnetite and an initial, predominantly multicore NP morphology. X-ray photoelectron spectroscopy (XPS) of these samples revealed that the surface of iron oxide NPs is enriched with Zn, although Zn species were also found deep under the iron oxide NP surface. For all the samples, XPS also demonstrates the atom ratio of Fe(3+)/Fe(2+) = 2:1, perfectly matching Fe3O4, but not ZnFe2O4, where Fe(2+) ions are replaced with Zn(2+). The combination of XPS with other physicochemical methods allowed us to propose that ZnO forms an ultrathin amorphous layer on the surface of iron oxide NPs and also diffuses inside the magnetite crystals. At higher Zn(acac)2 loading, cubic ZnO nanocrystals coexist with magnetite NPs, indicating a homogeneous nucleation of the former. The catalytic testing in syngas conversion to methanol demonstrated outstanding catalytic properties of Zn-containing magnetic oxides, whose activities are dependent on the Zn loading. Repeat experiments carried out with the best catalyst after magnetic separation showed remarkable catalyst stability even after five consecutive catalytic runs.

The synthesis and characterization of two high-valent vanadium-cyclo-P3 complexes, (nacnac)V(cyclo-P3)(Ntolyl2) (1) and (nacnac)V(cyclo-P3)(OAr) (2), and an inverted sandwich derivative, [(nacnac)V(Ntolyl2)]2(μ2-η(3):η(2)-cyclo-P3) (3), are presented. These novel complexes are prepared by activating white phosphorus (P4) with three-coordinate vanadium(II) precursors. Structural metrics, redox behavior, and DFT electronic structure analysis indicate that a [cyclo-P3](3-) ligand is bound to a V(V) center in monomeric species 1 and 2. A salient feature of these new cyclo-P3 complexes is their significantly downfield shifted (by ∼300 ppm) (31)P NMR resonances, which is highly unusual compared to related complexes such as (Ar[(i)Pr]N)3Mo(cyclo-P3) (4) and other cyclo-P3 complexes that display significantly upfield shifted resonances. This NMR spectroscopic signature was thus far thought to be a diagnostic property for the cyclo-P3 ligand related to its acute endocyclic angle. Using DFT calculations, we scrutinized and conceptualized the origin of the unusual chemical shifts seen in this new class of complexes. Our analysis provides an intuitive rational paradigm for understanding the experimental (31)P NMR spectroscopic signature by relating the nuclear magnetic shielding with the electronic structure of the molecule, especially with the characteristics of metal-cyclo-P3 bonding.

Incomplete nitrogen atom transfer from the iron(IV) nitride complex PhB(MesIm)3Fe≡N to the vanadium(III) complex V(Mes)3(THF) quantitatively provides the bimetallic complex PhB(MesIm)3Fe-N═V(Mes)3. Structural and spectroscopic characterizations reveal that the nitride ligand forms a linear bridge between V(V) and high-spin Fe(II) metal ions, confirming that atom transfer is accompanied by electron transfer. In the presence of an applied dc field, the complex displays slow relaxation of the magnetization, revealing its single-molecule magnet properties with an estimation of the energy barrier at about 10 K. This complex establishes a synthetic principle for the assembly of paramagnetic complexes bridged by nitride ligands.

Thermolysis of the iron(IV) nitride complex [PhB(tBuIm)3Fe≡N] with styrene leads to formation of the high-spin iron(II) aziridino complex [PhB(tBuIm)3Fe-N(CH2CHPh)]. Similar aziridination occurs with both electron-rich and electron-poor styrenes, while bulky styrenes hinder the reaction. The aziridino complex [PhB(tBuIm)3Fe-N(CH2CHPh)] acts as a nitride synthon, reacting with electron-poor styrenes to generate their corresponding aziridino complexes, that is, aziridine cross-metathesis. Reaction of [PhB(tBuIm)3Fe-N(CH2CHPh)] with Me3SiCl releases the N-functionalized aziridine Me3SiN(CH2CHPh) while simultaneously generating [PhB(tBuIm)3FeCl]. This closes a synthetic cycle for styrene azirdination by a nitride complex. While the less hindered iron(IV) nitride complex [PhB(MesIm)3Fe≡N] reacts with styrenes below room temperature, only bulky styrenes lead to tractable aziridino products., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cooperative binding of ion pairs to receptors is crucial for the manipulation of salts, but a comprehensive understanding of cooperativity has been elusive. To this end, we combine experiment and theory to quantify ion-pair binding and to separate allostery from electrostatics to understand their relative contributions. We designed aryl-triazole-ether macrocycles (MC) to be semiflexible, which allows ion pairs (NaX; X = anion) to make contact, and to be monocyclic to simplify analyses. A multiequilibrium model allows us to quantify, for the first time, the experimental cooperativity, α, for the equilibrium MC·Na(+) + MC·X(-) ⇌ MC·NaX + MC, which is associated with contact ion-pair binding of NaI (α = 1300, ΔGα = -18 kJ mol(-1)) and NaClO4 (α = 400, ΔGα = -15 kJ mol(-1)) in 4:1 dichloromethane-acetonitrile. We used accurate energies from density functional theory to deconvolute how the electrostatic effects and the allosteric changes in receptor geometry individually contribute to cooperativity. Computations, using a continuum solvation model (dichloromethane), show that allostery contributes ∼30% to overall positive cooperativity. The calculated trend of electrostatic cooperativity using pairs of spherical ions (NaCl > NaBr > NaI) correlates to experimental observations (NaI > NaClO4). We show that intrinsic ionic size, which dictates charge separation distance in contact ion pairs, controls electrostatic cooperativity. This finding supports the design principle that semiflexible receptors can facilitate optimal electrostatic cooperativity. While Coulomb's law predicts the size-dependent trend, it overestimates electrostatic cooperativity; we suggest that binding of the individual anion and cation to their respective binding sites dilutes their effective charge. This comprehensive understanding is critical for rational designs of ion-pair receptors for the manipulation of salts.

Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme required for hydroxylation of the aminobutyl side chain of deoxyhypusine in eukaryotic translation initiation factor 5A (eIF-5A), the second step in hypusine biosynthesis. DOHH has been recently identified in P. falciparum and P. vivax. Both enzymes have very peculiar features including E-Z type HEAT-like repeats and a diiron centre in their active site. Both proteins share only 26 % amino acid identity to the human paralogue. Hitherto, no X-ray structure exists from either enzyme. However, structural predictions based on the amino acid sequence of the active site in comparison to the human enzyme show that four conserved histidine and glutamate residues provide the coordination sites for chelating the ferrous iron ions. Recently, we showed that P. vivax DOHH is inhibited by zileuton (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea), a drug that is known for inhibiting human 5-lipoygenase (5-LOX) by the complexation of ferrous iron. A novel discovery program was launched to identify inhibitors of the P. falciparum DOHH from the Malaria Box, consisting of 400 chemical compounds, which are highly active in the erythrocytic stages of Malaria infections. In a first visual selection for potential ligands of ferrous iron, three compounds from different scaffold classes namely the diazonapthyl benzimidazole MMV666023 (Malaria Box plate A, position A03), the bis-benzimidazole MMV007384 (plate A, position B08), and a 1,2,5,-oxadiazole MMV665805 (plate A, position C03) were selected and subsequently evaluated in silico for their potential to complex iron ions. As a proof of principle, a bioanalytical assay was performed and the inhibition of hypusine biosynthesis was determined by GC-MS. All tested compounds proved to be active in this assay and MMV665805 exhibited the strongest inhibitory effect. Notably, the results were in accordance with the preliminary quantum-mechanical calculations suggesting the strongest iron complexation capacity for MMV665805. This compound might be a useful tool as well as a novel lead structure for inhibitors of P. falciparum DOHH.

In this study we enumerate the reactivity for two molecular vanadium nitrido complexes of [(nacnac)V≡N(X)] formulation [nacnac = (Ar)NC(Me)CHC(Me)(Ar)(-), Ar = 2,6-(CHMe2)2C6H3); X(-) = OAr (1) and N(4-Me-C6H4)2 (Ntolyl2) (2)]. Density functional theory calculations and reactivity studies indicate the nitride motif to have nucleophilic character, but where the nitrogen atom can serve as a conduit for electron transfer, thus allowing the reduction of the vanadium(V) metal ion with concurrent oxidation of the incoming substrate. Silane, H2SiPh2, readily converts the nitride ligand in 1 into a primary silyl-amide functionality with concomitant two-electron reduction at the vanadium center to form the complex [(nacnac)V{N(H)SiHPh2}(OAr)] (3). Likewise, addition of the B-H bond in pinacolborane to the nitride moiety in 2 results in formation of the boryl-amide complex [(nacnac)V{N(H)B(pinacol)}(Ntolyl2)] (4). In addition to spectroscopic data, complexes 3 and 4 were also elucidated structurally by single-crystal X-ray diffraction analysis. One-electron reduction of 1 with 0.5% Na/Hg on a preparative scale allowed for the isolation and structural determination of an asymmetric bimolecular nitride radical anion complex having formula [Na]2[(nacnac)V(N)(OAr)]2 (5), in addition to room-temperature solution X-band electron paramagnetic resonance spectroscopic studies.

The easy-to-prepare Nb(V) aryloxide complex [(ArO)2Nb(μ-Cl)Cl2]2 (OAr = 2,6-bis(diphenylmethyl)-4-tert-butylphenoxide) is a precursor to both Nb(IV), [trans-(ArO)2NbCl2(THF)2], and Nb(III), K3[(ArO)4Nb2(μ-Cl)3Cl2], molecules. The Nb(IV) and (V) complexes readily split atmospheric nitrogen at room temperature and 1 atmosphere, under reducing conditions, to produce the low-coordinate nitride dimer [(ArO)2Nb(μ-N)]2 and its radical anion, K[(ArO)2Nb(μ-N)]2.

A proteomic analysis of the interaction among multiprotein complexes involved in 2,3,7,8-dibenzo-p-dioxin (TCDD)-mediated toxicity in urinary bladder epithelial RT4 cells was performed using two-dimensional blue native SDS-PAGE (2D BN/SDS-PAGE). To enrich the protein complexes, unexposed and TCDD-exposed cells were fractionated. BN/SDS-PAGE of the resulting fractions led to an effective separation of proteins and protein complexes of various origins, including cell membrane, mitochondria, and other intracellular compartments. Major differences between the proteome of control and exposed cells involved the alteration of many calcium-regulated proteins (calmodulin, protein S100-A2, annexin A5, annexin A10, gelsolin isoform b) and iron-regulated proteins (ferritin, heme-binding protein 2, transferrin). On the basis of these findings, the intracellular calcium concentration was determined, revealing a significant increase after 24 h of exposure to TCDD. Moreover, the concentration of the labile iron pool (LIP) was also significantly elevated in TCDD-exposed cells. This increase was strongly inhibited by the calmodulin (CaM) antagonist W-7, which pointed toward a possible interaction between iron and calcium signaling. Because nitric oxide (NO) production was significantly enhanced in TCDD-exposed cells and was also inhibited by W-7, we hypothesize that alterations in calcium and iron homeostasis upon exposure to TCDD may be linked through NO generated by CaM-activated nitric oxide synthase. In our model, we propose that NO produced upon TCDD exposure interacts with the iron centers of iron-regulatory proteins (IRPs) that modulate the alteration of ferritin and transferrin, resulting in an augmented cellular LIP and, hence, increased toxicity.

We report a novel method for development of magnetically recoverable catalysts prepared by thermal decomposition of palladium acetylacetonate in the presence of iron oxide nanoparticles (NPs). Depending on conditions, the reaction results either in a dispersed mixture of Pd and iron oxide NPs or in their aggregates. It was demonstrated that the Pd loading, reaction temperature, solvent, and iron oxide NP size and composition are crucial to control the reaction product including the degree of aggregation of Pd and iron oxide NPs, and the catalyst properties. The aggregation controlled by polarization and magnetic forces allows faster magnetic separation, yet the aggregate sizes do not exceed a few hundred nanometers, making them suitable for various catalytic applications. These NP mixtures were studied in a selective hydrogenation of 2-methyl-3-butyn-2-ol to 2-methyl-3-buten-2-ol, demonstrating clear differences in catalytic behavior depending on the catalyst structure. In addition, one of the catalysts was also tested in hydrogenation of 3-methyl-1-pentyn-3-ol and 3-methyl-1-nonyn-3-ol, indicating some specificity of the catalyst toward different alkyne alcohols.

Fucosyltransferase VII encoded by the gene Fut7 is essential in CD4(+) T cells for the generation of E- and P-selectin ligands (E- and P-lig) which facilitate recruitment of lymphocytes into inflamed tissues and into the skin. This study aimed to identify regulatory elements controlling the inducible Fut7 expression in CD4(+) T cells that occurs upon activation and differentiation of naive T cells into effector cells. Comparative analysis of the histone modification pattern in non-hematopoetic cells and CD4(+) T cell subsets revealed a differential histone modification pattern within the Fut7 locus including a conserved non-coding sequence (CNS) identified by cross-species conservation comparison suggesting that regulatory elements are confined to this region. Cloning of the CNS located about 500 bp upstream of the Fut7 locus, into a luciferase reporter vector elicited reporter activity after transfection of the αβ-WT T cell line, but not after transfection of primary murine CD4(+) Th1 cells. As quantification of different Fut7 transcripts revealed a predominance of transcripts lacking the first exons in primary Th1 cells we searched for an alternative promoter. Cloning of an intragenic region spanning a 1kb region upstream of exon 4 into an enhancer-containing vector indeed elicited promoter activity. Interestingly, also the CNS enhanced activity of this intragenic minimal promoter in reporter assays in primary Th1 cells suggesting that both elements interact in primary CD4(+) T cells to induce Fut7 transcription., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The first highly diastereo- and enantioselective additions of a halogen and phosphoramidic acid to unactivated alkenes have been developed, catalyzed by a chiral Brønsted acid. A unique feature of these additions is the opportunity for stereocontrol at two noncontiguous chiral centers, carbon and phosphorus, leading to cyclic P-chiral phosphoramidates. In addition to their inherent value, the phosphoramidates are precursors to enantioenriched epoxy allylamines.

Memory type 1 T helper (T(H)1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the IFNG locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2Chi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the IFNG conserved non-coding sequence (CNS) 1, similar to memory CD8(+) T cells or T(H)1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2C(hi) NK cells. Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.

We describe synthesis and characterization of a derivative of aza-m-xylylene, diradical 2, that is persistent in solution at room temperature with the half-life measured in minutes (∼80-250 s) and in which the triplet ground state is below the lowest singlet state by >10 kcal mol(-1). The triplet ground states and ΔEST of 2 in glassy solvent matrix are determined by a new approach based on statistical analyses of their EPR spectra. Characterization and analysis of the analogous diradical 1 are carried out for comparison. Statistical analyses of their EPR spectra reliably provide improved lower bounds for ΔEST (from >0.4 to >0.6 kcal mol(-1)) and are compatible with a wide range of relative contents of diradical vs monoradical, including samples in which the diradical and monoradical are minor and major components, respectively. This demonstrates a new powerful method for the determination of the triplet ground states and ΔEST applicable to moderately pure diradicals in matrices.

The dimethyl aryloxide complexes [(PNP)M(CH3)2(OAr)] (M=Zr or Hf; PNP(-)=N[2-P(CHMe2)2-4-methylphenyl]2); Ar=2,6-iPr2C6H3), which were readily prepared from [(PNP)M(CH3)3] by alcoholysis with HOAr, undergo photolytically induced α-hydrogen abstraction to cleanly produce complexes [(PNP)M=CH2(OAr)] with terminal methylidene ligands. These unique systems have been fully characterized, including the determination of a solid-state structure in the case of M=Zr., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

East Coast fever (ECF) is a tick-borne disease caused by the parasite Theileria parva which infects cattle. In Sub-Saharan Africa it leads to enormous economic costs. After a bite of a tick, sporozoites invade the host lymphocytes and develop into schizonts. At this stage the parasite transforms host lymphocytes resulting in the clonal expansion of infected lymphocytes. Animals develop a lymphoma like disorder after infection which is rapidly fatal. Hitherto, a few drugs of the quinone type can cure the disease. However, therapy can only be successful after early diagnosis. The genera Theileria and Plasmodium, which includes the causative agent of human malaria, are closely related apicomplexan parasites. Enzymes of the hypusine pathway, a posttranslational modification in eukaryotic initiation factor EIF-5A, have shown to be druggable targets in Plasmodium. We identified the first enzyme of the hypusine pathway from T. parva, the deoxyhypusine synthase (DHS), which is located on chromosome 2 of the Muguga strain. Transcription is significantly increased in schizonts. The expressed T. parva DHS reveals an open reading frame (ORF) of 370 amino acids after expression in Escherichia coli Rosetta cells with a molecular size of 41.26 kDa and a theoretical pI of 5.26. Screening of the Malaria Box which consists of 400 active compounds resulted in a novel heterocyclic compound with a guanyl spacer which reduced the activity of T. parva DHS to 45%. In sum, the guanyl residue seems to be an important lead structure for inhibition of Theileria DHS. Currently, more different guanyl analogues from the Malaria Box are tested in inhibitor experiments to determine their efficacy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Deprotonation of the parent titanium imido ((tBu)nacnac)Ti≡NH(Ntolyl2) ((tBu)nacnac(-) = [ArNC(t)Bu]2CH; Ar = 2,6-(i)Pr2C6H3) with KCH2Ph forms a rare example of a molecular titanium nitride as a dimer, {[K][((tBu)nacnac)Ti≡N(Ntolyl2)]}2. From the parent imido or nitride salt, the corresponding aluminylimido-etherate adduct, ((tBu)nacnac)Ti≡N[AlMe2(OEt2)](Ntolyl2), can be isolated and structurally characterized. The parent imido is also a source for the related borylimido, ((tBu)nacnac)Ti═NBEt2(Ntolyl2).

Epidemiological studies suggest that environmental exposure to airborne particulate matter may promote cardiovascular diseases; however, it is not clear whether this observation actually reflects exposure to nanosized particles in the environment. In the present study, the human endothelial cell line EA.hy926 was exposed to pure carbon black and, to mimic exposure to diesel exhaust, carbon black loaded with benzo[a]pyrene to ascertain effects of these particles on the cell proteome and metabolom. Particular emphasis was laid on an extended exposure period (14 days) and a low particle concentration (100 ng/mL). While ROS production essentially remained unaffected, exposure of the cells to the particles resulted in a significantly enhanced cell proliferation. Evaluation of the obtained proteomic and phosphoproteomic data revealed modulations of proteins involved in catalytic processes and cytoskeleton maintenance. The bioinformatic evaluation of the data revealed the possible involvement of the transcription factor peroxisome proliferator-activated receptor gamma. The further analysis of the cytoskeleton indicated changes of the cell motility, which is in agreement with an observed increase in the cellular migration and invasion, and macroscopic changes of the cytoskeleton of the exposed cells.

A new pincer ligand is synthesized to be dianionic, with the potential to be redox active. It has pyrrrole rings attached to both ortho sites of a pyridine, as the linking element. This H2L can be doubly deprotonated and then used to replace two chloride ligands in MCl2(NCPh)2, to form LM(NCPh) for M = Pd, Pt. The acid form H2L reacts with ZnEt2 with elimination of only 1 mol of ethane to yield (HL)ZnEt, a three-coordinate species with one pendant pyrrole NH functionality. This molecule binds the Lewis base p-dimethylaminopyridine (DMAP) to give first a simple 1:1 adduct that eliminates ethane on heating to form four-coordinate LZn(DMAP), which has an unusual structure due to the strong preference of the pincer ligand to bind in a mer (planar) geometry. A molecule with two HL(-) ligands each bonded in a bidentate manner to FeCl2 is synthesized and shown to contain four-coordinate iron with a flattened-tetrahedral structure. The electrochemistry of LM(NCPh) and (L)Zn(DMAP) shows three oxidation processes, which is interpreted to involve at least two oxidations of the pyrrolide arms.

Aminyl tetraradicals with planar tetraazanonacene backbones have quintet (S = 2) ground states and do not show any detectable thermal population of the low-spin excited states up to the highest temperature investigated (100 K) in the 2-methyltetrahydrofuran (2-MeTHF) matrix. This indicates that the nearest electronic excited state (triplet) is at least ~0.3 kcal mol(-1) higher in energy, that is, the triplet-quintet energy gap, ΔE(TQ) > 0.3 kcal mol(-1), which is consistent with the broken-symmetry-DFT-computed ΔE(TQ) of about 5 kcal mol(-1). In concentrated (ca. 1-10 mM) solutions of tetraradical 4 in 2-MeTHF at 133 K, a fraction of tetraradicals form a dimer (association constant, K(assoc) ≈ 60 M(-1)), with a weak, antiferromagnetic exchange coupling, J/k ≈ -0.1 K ~ 0.2 cal mol(-1), between the S = 2 tetraradicals. This weak intradimer exchange coupling is expected for two tetraradicals at the distance of about 6 Å. The most sterically shielded tetraradical 5 in 2-MeTHF has a half-life of 1 h at room temperature; the product of its decay is the corresponding tetraamine, suggesting that the hydrogen atom abstraction from the solvent is primarily responsible for the decomposition of the tetraradical.

The bulky aryloxide 2,6-bis(diphenylmethyl)-4-tert-butylphenol [HOAr(tBu)] (1) can be synthesized from 4-tert-butylphenol and benzhydrol in solvent-free conditions and obtained pure in 91% yield. Deprotonation of HOAr(tBu) is accomplished with M(N(SiMe3)2) (M = Na, Li), yielding the corresponding salts of the aryloxide [MOAr(tBu)] (M(+) = Na (2), Li(3)) in 83% and 73% yield, respectively. Facile salt formation of the aryloxide ligand allows for transmetalation to a variety of metal halides. Through transmetalation reactions involving two aryloxides, mononuclear complexes of the type [M'(OAr(tBu))2Cl(THF)2] (M' = Sc (4), V (5), Cr (6), Ti (7)) can be prepared from the corresponding metal halide precursor MCl3(THF)3. Additionally, two aryloxides can be coordinated to Ti(IV) via a protonolysis route of Ti(NMe2)2Cl2 and 2 equiv of HOAr(tBu) to yield [Ti(OAr(tBu))2Cl2(NHMe2)] (8) in 72% isolated yield. Single-crystal X-ray diffraction studies of 1, 2, and the 3d metal complexes 5-8 clearly show the steric demand of the bulky ligand, whereas in transition metal complexes we do not observe the formation of mononuclear tris-aryloxide complexes.

Synthesis and characterization of divalent nickel complexed by 2-pyridylpyrrolide bidentate ligands are reported, as possible precursors to complexes with redox active ligands. Varied substituents on the pyrrolide, two CF3 (L(2)), two (t)Bu (L(0)), and one of each type (L(1)) are employed and the resulting Ni(L(n))2 complexes show different Lewis acidity toward CO, H2O, tetrahydrofuran (THF), or MeCN, the L(2) case being the most acidic. Density functional theory calculations show that the frontier orbitals of all three Ni(L(n))2 species are localized at the pyrrolide groups of both ligands and Ni(L(n))2(+) can be detected by mass spectrometry and in cyclic voltammograms (CVs). Following cyclic voltammetry studies, which show electroactivity primarily in the oxidative direction, reactions with pyridine N-oxide or Br2 are reported. The former yield simple bis adducts, Ni(L(2))2(pyNO)2 and the latter effects electrophilic aromatic substitution of the one pyrrolide ring hydrogen for both chelates, leaving it brominated.

The studies described in this paper deal with a sequence of cellular events induced by the environmental toxicant benzo[a]pyrene (B[a]P) that were investigated in primary urinary bladder epithelia cells (PUBEC) from pigs by using a proteomic approach. Two-dimensional (2DE) gel electrophoresis unveiled the differences in protein expression between cells exposed to 0.5 μM B[a]P for 24 h and control cells. Twenty-five differentially expressed proteins involved in DNA repair, mitochondrial dysfunction, and apoptosis were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). These findings were supported by the concentration-dependent increase in olive tail moments as determined by the comet assay and by a time-dependent increase in histone H2A.x (H2AX) phosphorylation upon B[a]P exposure. On the other hand, the expression of voltage-dependent anion channel 2 (VDAC2), cathepsin D (CTSD), heat shock protein 27 (HSP27), and heat shock protein 70 (HSP70) hinted to apoptosis occurring through the intrinsic apoptotic mitochondrial pathway. Taken together, these data suggest that B[a]P is capable of inducing DNA damage in urinary bladder epithelial cells at low concentrations during a short exposure period, thus eventually leading to cell death by apoptosis., Biological Significance: Epidemiological studies have indicated PAHs as potential candidates for initiating bladder cancer development, although the precise risk is still unknown (Kaufman et al. (2009)). In recent years, the understanding of the metabolic capacity of urothelial cells has broadened continuously; i.e. a wide range of xenobiotic metabolizing cytochrome P450 enzymes (CYP) were detected in urothelial cells from humans and animals (Roos et al., 2006; Guhe et al., 1996), thus indicating that urothelial cells are not only passively exposed to reactive metabolites but also actively by intracellularly producing reactive intermediates that can induce cancer. Moreover, small quantities of non-metabolized B[a]P and its hydroxylated derivatives have been identified in blood and urine (Rossella et al. (2009)). Thus, it appears plausible that B[a]P, a highly lipophilic compound, is taken up by the urothelium and metabolically activated to carcinogenic intermediates in these cells. In our previous studies with primary uroepithelial cells isolated from freshly slaughtered pigs we demonstrated the ability of these cells for a strong uptake of B[a]P and its conversion to the oxidative metabolite (3-OH-B[a]P) (Verma et al. (2012)). The present study is a continuation of this previous work exhibiting the effects of B[a]P exposure on cellular functions of PUBEC. The results indicated caspase-dependent apoptosis induced by B[a]P due to DNA damage (possibly lethal double-strand breaks as indicated by H2AX phosphorylation). Taken together, these studies provide strong evidence for the ability of B[a]P to act as a bladder carcinogen., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The potential redox activity of the 2,2'-pyridylpyrrolide ligand carrying two CF3 substituents (L(2)) is investigated. Synthesis and characterization of d(6) and d(7) species M(L(2))2 for M = Fe and Co are described (both are nonplanar, but not tetrahedral), as are the Lewis acidity of each. In spite of CV evidence for quasireversible reductions to form M(L(2))2(q-) where q = 1 and 2, chemical reductants instead yield divalent metal complexes KM(L(2))3, which show attractive interactions of K(+) to pyrrolide, to F, and to lattice toluene π cloud. The collected evidence on these products indicates that pyridylpyrrolide is a weak field ligand here, but CO can force spin pairing in Fe(L(2))2(CO)2. Evidence is presented that the overall reductive reaction yields 33 mol % of bulk metal, which is the fate of the reducing equivalents, and a mechanism for this ligand redistribution is proposed. Analogous ligand redistribution behavior is also seen for nickel and for trimeric monovalent copper analogues; reduction of Cu(L(2))2 simply forms Cu(L(2))2(-).

Dendritic cells from mesenteric lymph nodes (MLN) can convert retinal to retinoic acid (RA), which promotes induction of the gut-specific homing receptor α4β7. In contrast, priming within peripheral lymph nodes leads to upregulation of E- and P-selectin ligands (E- and P-lig). Apart from its α4β7 promoting effect, RA was shown to suppress E- and P-lig induction in vitro. However, enhanced frequencies of P-lig(+) CD4(+) T cells were reported during intestinal inflammation. To understand this contradiction, we first determined whether location of intestinal inflammation, that is, ileitis or colitis, affects P-lig induction. Both conditions promoted P-lig expression on CD4(+) T cells; however, P-lig expressed on T cells facilitated Th1 cell recruitment only into the inflamed colon but not into inflamed small intestine induced by oral Toxoplasma gondii infection. A majority of P-lig(+)CD4(+) T cells found within MLN during intestinal inflammation co-expressed α4β7 confirming their activation in the presence of RA. Mesenteric P-lig(+)CD4(+) cells co-expressed the 130 kDa isoform of CD43 which requires activity of core 2 (beta)1,6-N-acetyl-glycosaminyltransferase-I (C2GlcNAcT-I) suggesting that C2GlcNAcT-I contributes to P-lig expression under these conditions. To test whether inflammatory mediators can indeed overrule the inhibitory effect of RA on P-lig expression we stimulated CD4(+) T cells either polyclonal in the presence of IL-12 and IFNγ or by LPS-activated MLN-derived dendritic cells. Both conditions promoted P-lig induction even in the presence of RA. While RA impeded the induction of fucosyltransferase-VII it did not affect IL-12-dependent C2GlcNAcT-I induction suggesting that C2GlcNAcT-I can support P-lig expression even if fucosyltransferase-VII mRNA upregulation is dampened.

Complex (PNP)Ti=CH(t)Bu(CH(2)(t)Bu) (PNP(-) = N[2-P(CHMe(2))(2)-4-methylphenyl](2)) eliminates H(3)C(t)Bu to form transient (PNP)Ti≡C(t)Bu, which activates the C-F bond of ortho-difluoropyridine and ortho-fluoropyridine to form the alkylidene-fluoride complexes, (PNP)Ti=C[(t)Bu(NC(5)H(3)F)](F) (1) and (PNP)Ti=C[(t)Bu(NC(5)H(4))](F) (2), respectively. When (PNP)Ti=CH(t)Bu(CH(2)(t)Bu) is treated with meta-fluoropyridine, the ring-opened product (PNP)Ti(C((t)Bu)CC(4)H(3)-3-FNH) (3) is the only recognizable titanium metal complex formed. Theoretical studies reveal that pyridine binding disfavors 1,2-CF bond addition across the alkylidyne ligand in the case of ortho-fluoride pyridines, while sequential [2 + 2]-cycloaddition/β-fluoride elimination is a lower energy pathway. In the case of meta-fluoropyridine, [2 + 2]-cycloaddition and subsequent ring-opening metathesis is favored as opposed to C-H bond addition or sequential [2 + 2]-cycloaddition/β-hydride elimination. In all cases, C-H bond addition of ortho-fluoropyridines or meta-fluoropyridine is discouraged because such substrate must bind to titanium via its C-H bond, which is rather weak compared to the titanium-pyridine binding.

Thermal Bergman cyclization of Pt(II) dialkynylporphyrins reveals a marked reduction in the cyclization temperature relative to the free base and Zn(II) derivatives. In contrast, photogenerated (3)ππ* population produces no detectable Bergman photocyclization, suggesting that the photoreactivities of the related free base and Zn(II) derivatives occurs via the (1)ππ* state.

Reaction of 2 equiv of 1,2-bis((diphenylphosphino)ethynyl)benzene (dppeb, 1) with Pt(cod)Cl2 followed by treatment with N2H4 yields the reduced Pt(0) metalloenediyne, Pt(dppeb)2, 2. This complex is stable to both air oxidation and metal-mediated Bergman cyclization under ambient conditions due to the nearly idealized tetrahedral geometry. Reaction of 2 with 1 equiv of I2 in the presence of excess 1,4-cyclohexadiene (1,4-CHD) radical trap rapidly and near-quantitatively generates the cis-Bergman-cyclized, diiodo product 3 ((31)P: δ = 41 ppm, J(Pt-P) = 3346 Hz) with concomitant loss of 1 equiv of uncyclized phosphine chelate ((31)P: δ = -33 ppm). In contrast, addition of 2 equiv of I2 in the absence of additional radical trap instantaneously forms a metastable Pt(dppeb)2(2+) intermediate species, 4, that is characterized by δ = 51 ppm in the (31)P NMR (J(Pt-P) = 3171 Hz) and ν(C≡C) = 2169 cm(-1) in the Raman profile, indicating that it is an uncyclized, bis-ligated complex. Over 24 h, 4 undergoes ligand exchange to form a neutral, square planar complex that spontaneously Bergman cyclizes at ambient temperature to give the crystalline product Pt(dppnap-I2)I2 (dppnap-I2 = (1,4-diiodonaphthalene-2,3-diyl)bis(diphenylphosphine)), 5, in 52% isolated yield. Computational analysis of the oxidation reaction proposes two plausible flattened tetrahedral structures for intermediate 4: one where the phosphine core has migrated to a trans-spanning chelate geometry, and a second, higher energy structure (3.3 kcal/mol) with two cis-chelating phosphine ligands (41° dihedral angle) via a restricted alkyne-terminal starting point. While the energies are disparate, the common theme in both structures is the elongated Pt-P bond lengths (>2.4 Å), indicating that nucleophilic ligand substitution by I(-) is on the reaction trajectory to the cyclized product 5. The efficiency of the redox-mediated Bergman cyclization reaction of this stable Pt(0) metalloenediyne prodrug and resulting cisplatin-like byproduct represents an intriguing new strategy for potential dual-threat metalloenediyne therapeutics.

The recent introduction of the La(3+) precipitation method for the enrichment of phosphoproteins allows a gel-based analysis of these posttranslationally modified proteins. However, if this method is applied to cell lysates stored in urea-containing lysis buffer for an extended period of time, incomplete phosphoprotein recovery is observed. We ascribe this effect to the presence of urea in the lysis buffer. To overcome this problem various strategies were tested, where cell lysates stored at least for one year were utilized. By applying an optimized protocol approximately 250 proteins could be observed following separation by 2DE., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Serratezomine A is a member of the structurally diverse class of compounds known as the Lycopodium alkaloids. The key supporting studies and successful total synthesis of serratezomine A are described in this account. Significant features of the synthesis include the first application of free radical mediated vinyl amination and Hwu's oxidative allylation in a total synthesis and an intramolecular lactonization via a transannular S(N)i reaction. Minimal use of protecting groups and the highly diastereoselective formation of a hindered, quaternary stereocenter using an umpolung allylation are also highlights from a strategy perspective. Observation of quaternary carbon epimerization via a retro-Mannich/Mannich sequence highlights the additional challenge presented by the axial alcohol at C8 in serratezomine A.

Primaquine, an 8-aminoquinoline, is the only drug which cures the dormant hypnozoites of persistent liver stages from P. vivax. Increasing resistance needs the discovery of alternative pathways as drug targets to develop novel drug entities. Deoxyhypusine hydroxylase (DOHH) completes hypusine biosynthesis in eukaryotic initiation factor (eIF-5A) which is the only cellular protein known to contain the unusual amino acid hypusine. Modified EIF-5A is important for proliferation of the malaria parasite. Here, we present the first successful cloning and expression of DOHH from P. vivax causing tertiary malaria. The nucleic acid sequence of 1041 bp encodes an open reading frame of 346 amino acids. Histidine tagged expression of P. vivax DOHH detected a protein of 39.01 kDa in E. coli. The DOHH protein from P. vivax shares significant amino acid identity to the simian orthologues from P. knowlesi and P. yoelii strain H. In contrast to P. falciparum only four E-Z-type HEAT-like repeats are present in P. vivax DOHH with different homology to phycocyanin lyase subunits from cyanobacteria and in proteins participating in energy metabolism of Archaea and Halobacteria. However, phycocyanin lyase activity is absent in P. vivax DOHH. The dohh gene is present as a single copy gene and transcribed throughout the whole erythrocytic cycle. Specific inhibition of recombinant P. vivax DOHH is possible by complexing the ferrous iron with zileuton, an inhibitor of mammalian 5-lipoxygenase (5-LOX). Ferrous iron in the active site of 5-LOX is coordinated by three conserved histidines and the carboxylate of isoleucine(673). Zileuton inhibited the P. vivax DOHH protein with an IC50 of 12,5 nmol determined by a relative quantification by GC/MS. By contrast, the human orthologue is only less affected with an IC50 of 90 nmol suggesting a selective iron-complexing strategy for the parasitic enzyme.

The anilide-methyl complex (PNP)Sc(NH[DIPP])(CH(3)) (1) [PNP(-) = bis(2-diisopropylphosphino-4-tolyl)amide, DIPP = 2,6-diisopropylphenyl] eliminates methane (k(avg) = 5.13 × 10(-4) M(-1) s(-1) at 50 °C) in the presence of pyridine to generate the transient scandium imido (PNP)Sc═N[DIPP](NC(5)H(5)) (A-py), which rapidly activates the C-H bond of pyridine in 1,2-addition fashion to form the stable pyridyl complex (PNP)Sc(NH[DIPP])(η(2)-NC(5)H(4)) (2). Mechanistic studies suggest the C-H activation process to be second order overall: first order in scandium and first order in substrate (pyridine). Pyridine binding precedes elimination of methane, and α-hydrogen abstraction is overall-rate-determining [the kinetic isotope effect (KIE) for 1-d(1) conversion to 2 was 5.37(6) at 35 °C and 4.9(14) at 50 °C] with activation parameters ΔH(‡) = 17.9(9) kcal/mol and ΔS(‡) = -18(3) cal/(mol K), consistent with an associative-type mechanism. No KIE or exchange with the anilide proton was observed when 1-d(3) was treated with pyridine or thermolyzed at 35 or 50 °C. The post-rate-determining step, C-H bond activation of pyridine, revealed a primary KIE of 1.1(2) at 35 °C for the intermolecular C-H activation reaction in pyridine versus pyridine-d(5). Complex 2 equilibrated back to the imide A-py slowly, as the isotopomer (PNP)Sc(ND[DIPP])(η(2)-NC(5)H(4)) (2-d(1)) converted to (PNP)Sc(NH[DIPP])(η(2)-NC(5)H(3)D) over 9 days at 60 °C. Molecular orbital analysis of A-py suggested that this species possesses a fairly linear scandium imido motif (169.7°) with a very short Sc-N distance of 1.84 Å. Substituted pyridines can also be activated, with the rates of C-H activation depending on both the steric and electronic properties of the substrate.

More than 90 % of all bladder cancers are transitional cell carcinomas arising from the cells lining the inside of the hollow organ (uroepithelium). Cell cultures from primary urinary bladder epithelial cells (PUBEC) of pigs were established to assess the uptake, intracellular concentration, and subcellular distribution of the environmental pollutant benzo(a)pyrene (BaP). During treatment of the cells with 0.5 μM BaP for up to 24 h, intracellular concentration of BaP increased without saturation but with marked differences between various PUBEC pools. Analysis of BaP uptake by laser scanning microscopy indicated that BaP is rapidly partitioned into the cell membrane, while only a slight but significant increase in BaP fluorescence intensity was observed in the cytosol and nucleus. Spectrofluorometric quantification of BaP in PUBEC using ex situ calibration revealed a strong accumulation of BaP, leading to intracellular concentrations ranging from 7.28 to 35.70 μM in cells exposed to 0.5 μM BaP and from 29.9 to 406.64 μM in cells exposed to 10 μM BaP. These results were confirmed by gas chromatographic mass spectrometric analysis. Apoptotic cell nuclei were assessed by TUNEL analysis to see whether BaP exposure at the given concentrations results in a toxic effect. While apoptotic cells were barely detectable in control epithelial cells, there was a marked elevation in apoptosis in the BaP-exposed cells. In conclusion, a comprehensive study on uptake and quantification of BaP in epithelial cells from pig bladder is reported for the first time. The study may be helpful in understanding the pattern of BaP uptake and distribution in bladder and its possible implication in bladder cancer development.

Reduction of the dinuclear Co(II) nindigo complex dmp(2)Nin[Co(N{SiMe(3)}(2))](2), with 1 or 2 equiv. of K(0) (or KC(8)), affords the reduced complexes [dmp(2)Nin{Co(N{SiMe(3)}(2))}(2)](-) and [dmp(2)Nin{Co(N{SiMe(3)}(2))}(2)](2-), respectively. Inspection of these reduced species reveals ligand-centered reduction, with each cobalt ion retaining a formal 2+ oxidation state.

A series of sterically shielded pyrrolidine nitroxides were synthesized, and their reduction by ascorbate (vitamin C) indicate that nitroxide 3, a tetraethyl derivative of 3-carboxy-PROXYL, is reduced at the slowest rate among known nitroxides, i.e., at a 60-fold slower rate than that for 3-carboxy-PROXYL.

The synthesis and characterization of a series of iridium(I) and iridium(III) complexes supported by a 3,5-bis(trifluoromethyl)-2-(2'-pyridyl)pyrrole (L(-)) ligand are reported. Compound [Ir(L)(COD)] (COD = 1,5-cyclooctadiene) demonstrates reversible binding of hydrogen to form a dihydride complex [Ir(L)(H)(2)(COD)] with no hydrogenation of COD.

The air stable complex [(PNP)FeCl(2)] (1) (PNP = N[2-P(CHMe(2))(2)-4-methylphenyl](2)(-)), prepared from one-electron oxidation of [(PNP)FeCl] with ClCPh(3), displays an unexpected S = 3/2 to S = 5/2 transition above 80 K as inferred by the dc SQUID magnetic susceptibility measurement. The ac SQUID magnetization data, at zero field and between frequencies 10 and 1042 Hz, clearly reveal complex 1 to have frequency dependence on the out-of-phase signal and thus being a single molecular magnet with a thermally activated barrier of U(eff) = 32-36 cm(-1) (47-52 K). Variable-temperature Mössbauer data also corroborate a significant temperature dependence in δ and ΔE(Q) values for 1, which is in agreement with the system undergoing a change in spin state. Likewise, variable-temperature X-band EPR spectra of 1 reveals the S = 3/2 to be likely the ground state with the S = 5/2 being close in energy. Multiedge XAS absorption spectra suggest the electronic structure of 1 to be highly covalent with an effective iron oxidation state that is more reduced than the typical ferric complexes due to the significant interaction of the phosphine groups in PNP and Cl ligands with iron. A variable-temperature single crystal X-ray diffraction study of 1 collected between 30 and 300 K also reveals elongation of the Fe-P bond lengths and increment in the Cl-Fe-Cl angle as the S = 5/2 state is populated. Theoretical studies show overall similar orbital pictures except for the d(z(2)) orbital, which has the most sensitivity to change in the geometry and bonding, where the quartet ((4)B) and the sextet ((6)A) states are close in energy.

Asymmetric synthesis of (-)-[9]helicene, as well as preparation of its lower homologues, completes the series of carbon-sulfur [5]-, [7]-, [9]-, and [11]helicenes. Spectroscopic and electrochemical studies of this series provide an absorption onset-based band gap, E(g) = 3.40 eV, for a cross-conjugated (C(2)S)(n) helix; this value may be compared to E(g) = 3.59 eV obtained from TD-DFT computed excitation energies for a series of dimethyl-substituted [n]helicenes (n ≤ 31).

Benzo[a]pyrene (BaP), a five-ring polycyclic aromatic hydrocarbon, is a well-recognized environmental pollutant. Coal-processing waste products, petroleum sludge, asphalt, creosote, and tobacco smoke, all contain high levels of BaP. Exposure to BaP elicits many adverse biological effects, including tumor formation, immunosuppression, teratogenicity, and hormonal effects. In addition to the genetic damage caused by BaP exposure, several studies have indicated the disruption of protein-protein signaling pathways. However, contrary to the large number of studies on BaP-induced DNA damage, only few data have been gathered on its effects at the protein level. This review highlights all proteomic studies to date used for assessing the toxicity of BaP and its metabolites in various organ systems. It will also give an overview on the role proteomics may play to elucidate the mechanisms underlying BaP toxicity., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The title compound, C(16)H(16)N(2)O(2)S(2), has been synthesized by several different high-yield routes, and has been encountered as a co-product in a number of reaction pathways, ever since it became of inter-est to our research program over 30 years ago. We now confirm the proposed mol-ecular structure in which the mol-ecule exhibits a twofold axis of symmetry through the mid-point of the S-S bond and the two planes defined by the (carbamo-yl)sulfenyl moieties are essentially perpendicular to each other [dihedral angle = 81.55 (14)°].

We describe the synthesis and electronic properties of ladder oligomers of poly(m-aniline) that may be considered as derivatives of azaacenes with cross-conjugated π-systems. Syntheses of ladder oligo(m-aniline)s with 9 and 13 collinearly fused six-membered rings employed Pd-catalyzed aminations and Friedel-Crafts-based ring closures. Structures were confirmed by either X-ray crystallography or correlations between DFT-computed and experimental spectroscopic data such as (1)H, (13)C, and (15)N NMR chemical shifts and electronic absorption spectra. All compounds have planar "azaacene" moieties. The experimental band gaps E(g) ≈ 3.5-3.65 eV, determined by the UV-vis absorption onsets, were in agreement with the TD-DFT-computed vertical excitation energies to the S(1) state. Fluorescence quantum yields of up to 20% were found. Electrochemically estimated HOMO energies of -4.8 eV suggested propensity for a facile one-electron oxidation and just sufficient environmental stability toward oxygen (O(2)). For two oligomers with "tetraazanonacene" moieties, potentials of E(4+/3+) ≈ 1.6-1.7 V vs SCE were determined for four-electron oxidation to the corresponding tetraradical tetracations.

Treatment of d(1) [(nacnac)TiCl(Ntol(2))] with NaN(3) results in NaCl formation and N(2) ejection to yield the first four coordinate, parent imide [(nacnac)Ti=NH(Ntol(2))] (nacnac(-)=[ArNC(CH(3))](2)CH, Ar = 2,6-iPr(2)C(6)H(3), tol = 4-CH(3)C(6)H(4))., (This journal is © The Royal Society of Chemistry 2012)

Posttranslational modification (PTM) of proteins, particularly phosphorylation, is a key element in the regulation of cell functions. In many signal transduction processes, PTM is a pivotal step. Various analytical methods have been proposed for the identification of phosphoproteins; however, most of these methods require sophisticated equipment. Here we present an easily applicable method of phosphoprotein enrichment. This method is based on single-step precipitation by lanthanum chloride and allows subsequent protein identification by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF-MS). The method proved its suitability for the isolation of phosphoproteins from frozen tissue and cultured cells samples after cell lysis in various buffer systems (urea/thiourea and EGTA/EDTA). The tests revealed that the isolation of phosphoproteins can be achieved with high efficiency even from complex protein mixtures. Our results indicate that lanthanum-based enrichment of phosphoproteins can be a useful tool in phosphoproteomic studies., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The synthesis of a new series of free-base, Ni(II) and Zn(II) 2,3,12,13-tetra(ethynyl)-5,10,15,20-tetraphenyl porphyrins is described. Upon heating, two of the four ethynyl moieties undergo Bergman cyclization to afford the monocyclized 2,3-diethynyl-5,20-diphenylpiceno[10,11,12,13,14,15-jklmn]porphyrin in 30 %, 10 %, and trace yields, respectively. The structures of all products were investigated by using quantum chemical calculations and the free-base analogue was isolated and crystallized; all compounds show significant deviation from the idealized planar structure. No fully-cyclized bispiceno[20,1,2,3,4,5,10,11,12,13,14,15-fghij]porphyrin was isolated from the reaction mixture. To understand why only two of the four enthynyl groups undergo Bergman cyclization, the reaction coordinates were examined by using DFT at the PWPW91/cc-pVTZ(-f) level coupled to a continuum solvation model. The barrier to cyclization of the second pair of ethynyl groups was found to be 5.5 kcal  mol(-1) higher than the first, suggesting a negative cooperative effect and significantly slower rate for the second cyclization. Cyclization reactions for model porphyrin-enediynes with ethene- and H-functionality substitutions at the meso-phenyl rings were also examined, and found to have a similar barrier to diradical formation for the second cyclization event as for the first in these highly planar molecules. By enforcing an artificial 30° cant in two of the pyrrole rings of the porphyrin, the second barrier was increased by 2 kcal  mol(-1) in the ethene model system; this suggests that the disruption of the π conjugation of the extended porphyrin structure is the cause of the increased barrier to the second cyclization event., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Epithelial tissue lining the inner side of the urinary bladder is the most common target for bladder cancer-related diseases. Bladders of freshly slaughtered pigs were utilised for a comprehensive analysis of the proteome and phosphoproteome of bladder epithelial cells. Following protein separation by 2-D gel electrophoresis and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) the first proteome and phosphoproteome maps of pig urinary bladder epithelial cells (PUBEC) were established. A total of 120 selected protein spots were identified. By using the La(3+) enrichment method further developed in our laboratory we identified 31 phosphoproteins with minimal contamination by non-phosphopeptides. The 2-DE map of pig urothelial cells may prove as a useful tool for studies on uroepithelial biology, and the analysed phosphoproteins expression pattern, together with the whole cell proteome, will be helpful for identifying the proteins involved in bladder-related diseases., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Excessive orbital fibroblast (OF) proliferation and extracellular matrix production, as well as inflammation resulting in the expansion and remodeling of orbital tissue, are characteristic of Graves ophthalmopathy (GO). Our aim was to analyze and inhibit signaling pathways in resident OF that are involved in GO. METHODS/MAIN OUTCOME MEASURES: Primary human OF were obtained from 12 patients with active, severe GO and from 12 healthy control subjects. The cells were characterized by immunofluorescence assay and flow cytometry. Tyrosine phosphorylation of cellular proteins was determined by Western blot techniques, immunoprecipitation, and protein identity with mass spectrometry. Cell proliferation was determined by 5-bromo-2-deoxyuridine incorporation, hyaluronan (HA) production was assessed by a HA-binding protein based assay, and intracellular reactive oxygen species (ROS) were determined by the dichlorofluorescein assay. Clathrin heavy-chain (CHC) expression was inhibited with small interfering RNA technology., Results: Tyrosine phosphorylation of CHC is constitutively increased in vitro in GO-derived OF, independent of serum or other stimulating factors. The proliferative and biosynthetic capabilities (production of HA, ROS) of GO-derived OF are significantly higher than those of OF from healthy control subjects. Down-regulation of CHC expression leads to a normalization of pathologically increased proliferation and production of HA and ROS in GO-derived OFs in vitro., Conclusions: Our findings strongly suggest that clathrin and clathrin-mediated signaling pathways are involved in the inflammatory signal transduction of OF in GO. With the identification of clathrin, we report a new potential targeting molecule for specific pharmacological inhibition of the local inflammatory response characteristic of GO.

Oxidation of diaryldiamine 2, a tetrahydrodiazapentacene derivative, provides diarylnitroxide diradical 1 accompanied by an intermediate nitroxide monoradical and a multitude of isolable diamagnetic products. DFT-computed tensors for EPR spectra and paramagnetic (1)H NMR isotropic shifts for nitroxide diradical 1 show good agreement with the experimental EPR spectra in rigid matrices and paramagnetic (1)H NMR spectra in solution, respectively. Examination of the diamagnetic products elucidates their formation via distinct pathways involving C-O bond-forming reactions, including Baeyer-Villiger-type oxidations. An unusual diiminoketone structure and two spirocyclic structures of the predominant diamagnetic products are confirmed by either X-ray crystallography or correlations between DFT-computed and experimental spectroscopic data such as (1)H, (13)C, and (15)N NMR chemical shifts and electronic absorption spectra.

The addition of 2,6-dimethylphenyl isocyanide (CN[DMeP], two equivalents) to previously reported (PNP)Sc(III) pyridyl complexes resulted in the formation of novel indoline complexes of scandium. By varying the nature of the pyridyl moiety one can intercept an intermediate prior to methyl migration. In addition to structural studies of two indolene complexes, we also propose a mechanism for the insertion and indolene ring closure., (This journal is © The Royal Society of Chemistry 2011)