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Fluorine labelling of a Ru(iii) chemotherapeutic candidate enables studies of reactivity in tissues by 19F nuclear magnetic resonance imaging methods.

Appending of ferrocene (Fc) to biologically-active organic backbones can generate novel multi-functional species for targeting bacteria and cancer. In this work Fc was linked to coumarin and anthraquinone with the goal of harnessing the redox-active Fc centre to generate new compounds that exhibit cytoxicity through the generation of toxic reactive oxygen species (ROS). A Cu(I)-catalyzed azide-alkyne cycloaddition "click" reaction was used to connect the organic and Fc components

The application of CF

The reduction of iron(II) phthalocyanine (Pc(2-)Fe

Targeting the low-oxygen (hypoxic) environments found in many tumours by using redox-active metal complexes is a strategy that can enhance efficacy and reduce the side effects of chemotherapies. We have developed a series of CuII complexes with tridentate pyridine aminophenolate-based ligands for preferential activation in the reduction window provided by hypoxic tissues. Furthermore, ligand functionalization with a pendant CF3 group provides a 19 F spectroscopic handle for magnetic-resonance studies of redox processes at the metal centre and behaviour in cellular environments. The phenol group in the ligand backbone was substituted at the para position with H, Cl, and NO2 to modulate the reduction potential of the CuII centre, giving a range of values below the window expected for hypoxic tissues. The NO2 -substituted complex, which has the highest reduction potential, showed enhanced cytotoxic selectivity towards HeLa cells grown under hypoxic conditions. Cell death occurs by apoptosis, as determined by analysis of the cell morphology. A combination of 19 F NMR and ICP-OES indicates localization of the NO2 complex in HeLa cell nuclei and increased cellular accumulation under hypoxia. This correlates with DNA nuclease activity being the likely origin of cytotoxic activity, as demonstrated by cleavage of DNA plasmids in the presence of the CuII nitro complex and a reducing agent. Selective detection of the paramagnetic CuII complexes and their diamagnetic ligands by 19 F MRI suggests hypoxia-targeting theranostic applications by redox activation.

The interplay between the primary and secondary coordination spheres in biological metal sites plays an essential role in controlling their properties. Some of the clearest examples of this are from copper sites in blue and purple copper proteins. Many such proteins contain methionine (Met) in the primary coordination sphere as a weakly bound ligand to Cu. While the effects of replacing the coordinated Met are understood, less so is the importance of its second-sphere interactions. In this combined informatics and experimental study, we first present a bioinformatics investigation of the second-sphere environments in biological Met-Cu motifs. The most common interaction is between the Met-CH

Significance Pyridoxal phosphate (PLP)-dependent enzymes rarely react with oxygen, but an emerging group of oxygen-, PLP-dependent enzymes oxidize l -arginine. Two types of oxidases are known: hydroxylases and desaturases. We demonstrate that arginine desaturases have a minor hydroxylase activity and then show through X-ray crystallographic, mutagenesis, spectroscopic, and computational studies that their mechanism involves two rounds of single-electron transfer to oxygen and superoxide rebound, ultimately giving a conjugated hydroperoxyl intermediate. Water can attack to give a hydroxylated product and release H 2 O 2 , but with water absent, the intermediate can be deprotonated, instead giving a desaturated product and H 2 O 2 . Our work outlines the unique mechanism and evolutionary history of these enzymes and sets the stage toward engineering these enzymes to catalyze oxidative reactions.

Alzheimer's disease (AD) is a multifaceted disease that is characterized by increased oxidative stress, metal-ion dysregulation, and the formation of intracellular neurofibrillary tangles and extracellular amyloid-β (Aβ) aggregates. In this work we report the large affinity binding of the iron(III) 2,17-bis-sulfonato-5,10,15-tris(pentafluorophenyl)corrole complex FeL1 to the Aβ peptide (Kd ∼ 10−7) and the ability of the bound FeL1 to act as a catalytic antioxidant in both the presence and absence of Cu(II) ions. Specific findings are that: (a) an Aβ histidine residue binds axially to FeL1; (b) that the resulting adduct is an efficient catalase; (c) this interaction restricts the formation of high molecular weight peptide aggregates. UV-Vis and electron paramagnetic resonance (EPR) studies show that although the binding of FeL1 does not influence the Aβ–Cu(II) interaction (Kd ∼ 10−10), bound FeL1 still acts as an antioxidant thereby significantly limiting reactive oxygen species (ROS) generation from Aβ-Cu. Overall, FeL1 is shown to bind to the Aβ peptide, and modulate peptide aggregation. In addition, FeL1 forms a ternary species with Aβ–Cu(II) and impedes ROS generation, thus showing the promise of discrete metal complexes to limit the toxicity pathways of the Aβ peptide.

Organometallic Ru(II)-cymene complexes linked to ferrocene (Fc) via nitrogen heterocycles have been synthesized and studied as cytotoxic agents. These compounds are analogues of Ru(II)-arene piano-stool anticancer complexes such as RAPTA-C. The Ru center was coordinated by pyridine, imidazole, and piperidine with 0-, 1-, or 2-carbon bridges to Fc to give six bimetallic, dinuclear compounds, and the properties of these complexes were compared with their non-Fc-functionalized parent compounds. Crystal structures for five of the compounds, their Ru-cymene parent compounds, and an unusual trinuclear compound were determined. Cyclic voltammetry was used to determine the formal MIII/II potentials of each metal center of the Ru-cymene-Fc complexes, with distinct one-electron waves observed in each case. The Fc-functionalized complexes were found to exhibit good cytotoxicity against HT29 human colon adenocarcinoma cells, whereas the parent compounds were inactive. Similarly, antibacterial activity from the Ru-cymene-Fc compounds was observed against Bacillus subtilis, but not from the unfunctionalized complexes. In both cases, the IC50 values correlated quantitatively with the Fc+/0 reduction potentials. This is consistent with more facile oxidation to give ferrocenium, and subsequent generation of toxic reactive oxygen species, leading to greater cytotoxicity. The antioxidant properties of the complexes were quantified by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. EC50 values indicate that linking of the Ru and Fc centers promotes antioxidant activity.

Low aqueous solubility is a major barrier to the clinical application of otherwise promising drug candidates. We demonstrate that this issue can be resolved in medicinal molecules containing potential ligating groups, through the addition of labile transition-metal ions. Incubation of the chemotherapeutic CX5461 with Cu2+ or Zn2+ enables solubilization at neutral pH but does not affect intrinsic cytotoxicity. Spectroscopic and computational studies demonstrate that this arises from coordination to the pyrazine functionality of CX5461 and may involve bidentate coordination at physiological pH.

A series of octahedral CoIII salen complexes (where salen represents a N2O2 bis-Schiff-base bis-phenolate framework) were prepared with axial imidazole ligating groups. When using 1-methylimidazole (1-MeIm) axial ligands, the CoIII/CoII reduction potential could be altered by 220 mV via variation of the electron-donating ability of the para-ring substituents (R = H (1), OMe (2), tBu (3), Br (4), NO2 (5), and CF3 (6)). In addition, the irreversibility of the reduction process suggested substantial geometrical changes and axial ligand exchange upon reduction to the more labile CoII oxidation state. Installing an imidazole-coumarin conjugate as the axial ligands resulted in fluorescence quenching when bound to the CoIII centre (R = H (7), OMe (8), and CF3 (9)). The redox properties and fluorescence increase upon ligand release for 7–9 were studied under reducing conditions and in the presence of excess competing ligand (1-MeIm). It was determined that the Lewis acidity of the CoIII centre was the dominant factor in controlling axial ligand exchange for this series of complexes.

Alzheimer's disease (AD) is the most common form of dementia, leading to loss of cognition, and eventually death. The disease is characterized by the formation of extracellular aggregates of the amyloid-beta (Aβ) peptide and neurofibrillary tangles of tau protein inside cells, and oxidative stress. In this study, we investigate a series of Ru(III) complexes (Ru-N) derived from NAMI-A in which the imidazole ligand has been substituted for pyridine derivatives, as potential therapeutics for AD. The ability of theRu-Nseries to bind to Aβ was evaluated by NMR and ESI-MS, and their influence on the Aβ peptide aggregation process was investigated via electrophoresis gel/western blot, TEM, turbidity, and Bradford assays. The complexes were shown to bind covalently to the Aβ peptide, likely via a His residue. Upon binding, the complexes promote the formation of soluble high molecular weight aggregates, in comparison to peptide precipitation for peptide alone. In addition, TEM analysis supports both amorphous and fibrillar aggregate morphology forRu-Ntreatments, while only large amorphous aggregates are observed for peptide alone. Overall, our results show that theRu-Ncomplexes modulate Aβ peptide aggregation, however, the change in the size of the pyridine ligand does not substantially alter the Aβ aggregation process.

A tridentate benzoxazole-containing aminophenol ligand NNOH2 was coordinated to Co and V metal centers and the electronic structure of the resultant complexes characterized by both experimental and theoretical methods. The solid state structure of the Co complex exhibits a distorted octahedral geometry with two tridentate ligands bound in meridional fashion, and coordination-sphere bond lengths consistent with a Co(III) oxidation state. EPR and magnetic data support a S = 1/2 ground state, and a formal electronic description of Co(III)(NNOAP)(NNOISQ) where NNOAP corresponds to an amidophenoxide and NNOISQ to the iminosemiquinone redox level. However, the metrical parameters are similar for both ligands in the solid state, and DFT calculations support delocalization of the ligand radical over both ligands, affording an intermediate ligand redox level Co(III)(NNO1.5−)(NNO1.5−). The vanadyl complex exhibits a distorted octahedral geometry in the solid state consistent with a V(V) metal center and amidophenoxide (NNOAP), acetylacetonate and oxo ligands. The ligand metrical parameters are consistent with significant amidophenoxide to V(V) π donation. Overall, our results highlight the roles of electron transfer, delocalization, and π bonding in the metal complexes under study, and thus the complexity in assignment of the electronic structure in these systems.

Electron paramagnetic resonance spectroscopy (EPR) is an invaluable method for characterizing ligand environments and oxidation states of paramagnetic metal complexes. In this review we describe applications of this technique to understanding the often complicated mechanisms of metal-based anticancer compounds. EPR has been instrumental in unravelling this in many instances, providing insight into conformations upon dissolution, aqueous ligand exchange, pH effects, and redox processes. EPR studies have also characterized the protein and DNA interactions of metal-based chemotherapeutics, enabling discrimination of different binding modes. EPR can also be used to probe the behaviour of metallodrugs with whole cells, revealing interactions with cellular components and redox stability. Together, these studies provide important information on aspects of anticancer activity and demonstrate the scope and potential of EPR to contribute to the ongoing development of metal-based anticancer complexes.

The Ru(III) complexes indazolium [trans-RuCl4(1H-indazole)2] (KP1019) and sodium [trans-RuCl4(1H-indazole)2] (NKP-1339) are leading candidates for the next generation of metal-based chemotherapeutics. Trifluoromethyl derivatives of these compounds and their imidazole and pyridine analogues were synthesized to probe the effect of ligand lipophilicity on the pharmacological properties of these types of complexes. Addition of CF3 groups also provided a spectroscopic handle for (19)F NMR studies of ligand exchange processes and protein interactions. The lipophilicities of the CF3-functionalized compounds and their unsubstituted parent complexes were quantified by the shake-flask method to give the distribution coefficient D at pH 7.4 (log D7.4). The solution behavior of the CF3-functionalized complexes was characterized in phosphate-buffered saline (PBS) using (19)F NMR, electron paramagnetic resonance (EPR), and UV-vis spectroscopies. These techniques, along with fluorescence competition experiments, were also used to characterize interactions with human serum albumin (HSA). From these studies it was determined that increased lipophilicity correlates with reduced solubility in PBS but enhancement of noncoordinate interactions with hydrophobic domains of HSA. These protein interactions improve the solubility of the complexes and inhibit the formation of oligomeric species. EPR measurements also demonstrated the formation of HSA-coordinated species with longer incubation. (19)F NMR spectra show that the trifluoromethyl complexes release axial ligands in PBS and in the presence of HSA. In vitro testing showed that the most lipophilic complexes had the greatest cytotoxic activity. Addition of CF3 groups enhances the activity of the indazole complex against A549 nonsmall cell lung carcinoma cells. Furthermore, in the case of the pyridine complexes, the parent compound was inactive against the HT-29 human colon carcinoma cell line but showed strong cytotoxicity with CF3 functionalization. Overall, these studies demonstrate that lipophilicity may be a determining factor in the anticancer activity and pharmacological behavior of these types of Ru(III) complexes.

Herein we describe the synthesis, structure and electronic properties of an unusual redox-active ditopic ligand with a stable open-shell configuration. This stable phenoxyl radical features intense and very low energy electronic transitions in the near infrared (NIR) part of the spectrum and is structurally set up to strongly spin couple coordinated transition metal ions in [2 × 2] grid-type structures.

The ruthenium anticancer compound NAMI-A, imidazolium [trans-RuCl4(1H-imidazole)(DMSO-S)], is currently undergoing advanced clinical evaluation. As with other Ru(iii) chemotherapeutic candidates, interactions with human serum albumin (HSA) have been identified as a key component of the speciation of NAMI-A following intravenous administration. To characterize coordination to HSA, we have performed electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic analysis of deuterium-labelled isotopologues of both NAMI-A and its bis-DMSO analogue, [(DMSO)2H][trans-RuCl4(DMSO-S)2] (Ru-bis-DMSO). Samples were prepared using phosphate buffered saline, in the presence of HSA, and with the individual amino acids histidine, cysteine, and alanine. Analysis of (1)H ENDOR spectra shows characteristic hyperfine interactions from DMSO, water, and imidazole ligands. Furthermore, coordination of imidazole ligands was confirmed from diagnostic (14)N ENDOR signals. Combined with the EPR data from the complexes following incubation in the presence of histidine, the ENDOR data demonstrate that both complexes bind to HSA via histidine imidazoles. Furthermore, the protein-bound species are shown to have water ligands and, in the case of Ru-bis-DMSO, one species has a remaining coordinated DMSO.

Adenosylcobalamin-dependent ornithine 4,5-aminomutase (OAM) from Clostridium sticklandii utilizes pyridoxal 5'-phosphate (PLP) to interconvert d-ornithine to 2,4-diaminopentanoate via a multistep mechanism that involves two hydrogen transfer steps. Herein, we uncover features of the OAM catalytic mechanism that differentiate it from its homologue, the more catalytically promiscuous lysine 5,6-aminomutase. Kinetic isotope effects (KIEs) with dl-ornithine-3,3,4,4,5,5-d6 revealed a diminished (D)kcat/Km of 2.5 ± 0.4 relative to a (D)kcat of 7.6 ± 0.5, suggesting slow release of the substrate from the active site. In contrast, a KIE was not observed on the rate constant associated with Co-C bond homolysis as this step is likely "gated" by the formation of the external aldimine. The role of tyrosine 187, which lies planar to the PLP pyridine ring, was also investigated via site-directed mutagenesis. The 25- and 1260-fold reduced kcat values for Y187F and Y187A, respectively, are attributed to a slower rate of external aldimine formation and a diminution of adenosylcobalamin Co-C bond homolysis. Notably, electron paramagnetic resonance studies of Y187F suggest that the integrity of the active site is maintained as cob(II)alamin and the PLP organic radical (even at lower concentrations) remain tightly exchange-coupled. Modeling of d-lysine and l-β-lysine into the 5,6-LAM active site reveals interactions between the substrate and protein are weaker than those in OAM and fewer in number. The combined data suggest that the level of protein-substrate interactions in aminomutases not only influences substrate specificity, but also controls radical chemistry.

A series of pyridine-based derivatives of the clinically successful Ru(III)-based complexes indazolium [trans-RuCl4(1H-indazole)2] (KP1019) and sodium [trans-RuCl4(1H-indazole)2] (KP1339) have been synthesized to probe the effect of hydrophobic interactions with human serum albumin (hsA) on anticancer activity. The solution behavior and protein interactions of the new compounds were characterized by using electron paramagnetic resonance (EPR) and UV/Vis spectroscopy. These studies have revealed that incorporation of hydrophobic substituents at the 4'-position of the axial pyridine ligand stabilizes non-coordinate interactions with hsA. As a consequence, direct coordination to the protein is inhibited, which is expected to increase the bioavailability of the complexes, thus potentially leading to improved anticancer activity. By using this approach, the lifetimes of hydrophobic protein interactions were extended from 2 h for the unsubstituted pyridine complex, to more than 24 h for several derivatives. Free complexes were tested for their anticancer activity against the SW480 human colon carcinoma cell line, exhibiting low cytotoxicity. Pre-treatment with hsA improved the solubility of every compound and led to some changes in activity. Particularly notable was the difference in activity between the methyl- and dibenzyl-functionalized complexes. The former shows reduced activity after incubation with hsA, indicating reduced bioavailability due to protein coordination. The latter exhibits little activity on its own but, following treatment with hsA, exhibited significant cytotoxicity, which is consistent with its ability to form non-coordinate interactions with the protein. Overall, our studies demonstrate that non-coordinate interactions with hsA are a viable target for enhancing the activity of Ru(III)-based complexes in vivo.

Rapid and low overpotential oxidation of water to dioxygen remains a key hurdle for storage of solar energy. Here, we address this issue by demonstrating that deprotonation of 2-(2′-pyridyl)-imidazole (pimH)-ligated copper complexes promotes water oxidation at low overpotential and low catalyst loading. This improves upon other work on homogeneous copper-based water oxidation catalysts, which are highly active, but limited by high overpotentials. EPR and UV-vis spectroscopic evaluation of catalyst speciation shows that at pH ≥ 12 coordinated pimH is deprotonated and a bis(hydroxide) Cu2+ active catalyst forms. Rapid electrochemical water oxidation (35 s−1, 0.85 V onset potential) was observed with 150 μM catalyst. These results demonstrate that catalytic water oxidation potentials can be shifted by hundreds of mV in homogeneous metal catalysts bearing an ionisable imidazole ligand.

The Cu(II) complex CuCl2(pbzH), pbzH=2-(2-pyridyl)benzimidazole, and derivatives modified at the non-coordinated nitrogen of the benzimidazole fragment, have been studied as anticancer agents. These compounds show promising cytotoxicity against A549 adenocarcinomic alveolar basal epithelial cells with IC50 values in the range of 5-10μM. Importantly, this activity is higher than either CuCl2·2H2O or the individual ligands, demonstrating that ligand coordination to the Cu(II) centres of the complexes is required for full activity. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopies were used to characterize the solution behaviour of the complexes. These studies demonstrate: (i) two types of solvated species in buffer, (ii) both coordinate and non-coordinate interactions with albumin, and (iii) weak interactions with DNA. Further DNA studies using agarose gel electrophoresis demonstrate strand cleavage by the complexes in the presence of ascorbate, which is mediated by reactive oxygen species (ROS). Through a fluorescence-based in vitro assay, intracellular ROS generation in the A549 cell line was observed; indicating that damage by ROS is responsible for the observed activity of the complexes.

In 1965, gold(II) phthalocyanine (AuPc, 1) was described to be synthesized from unsubstituted 1,3-diiminoisoindoline and gold powder or AuBr. Compound 1 has been regarded as a rare example of a paramagnetic gold(II) complex. However, its chemistry, especially the oxidation state of the central gold ion, has not been previously explored due to the inherent insolubility of 1 caused by its unsubstituted structure. In our attempt to synthesize soluble AuPcs by using 5,6-di-substituted 1,3-diiminoisoindolines, gold(III) phthalocyanine chloride (3) and a gold(III) complex of an unprecedented ring-contracted phthalocyanine analogue ([18]tribenzo-pentaaza-triphyrin(4,1,1), 4) were isolated. With this discrepant result from the original literature in hand, a reinvestigation of the original AuPc synthesis by using unsubstituted 1,3-diiminoisoindoline and various gold salts (including gold powder and AuBr) was performed, finding that only unsubstituted analogues of 3 and 4 or free-base phthalocyanine were obtained. No gold(II)-containing species could be isolated.

A series of pyridine-based derivatives of the antimetastatic Ru(III) complex imidazolium [trans-RuCl(4)(1H-imidazole)(DMSO-S)] (NAMI-A) have been synthesized along with their sodium-ion compensated analogues. These compounds have been characterized by X-ray crystallography, electron paramagnetic resonance (EPR), NMR, and electrochemistry, with the goal of probing their noncovalent interactions with human serum albumin (hsA). EPR studies show that the choice of imidazolium ligands and compensating ions does not strongly influence the rates of ligand exchange processes in aqueous buffer solutions. By contrast, the rate of formation and persistence of interactions of the complexes with hsA is found to be strongly dependent on the properties of the axial ligands. The stability of noncovalent binding is shown to correlate with the anticipated ability of the various pyridine ligands to interact with the hydrophobic binding domains of hsA. These interactions prevent the oligomerization of the complexes in solution and limit the rate of covalent binding to albumin amino acid side chains. Electrochemical studies demonstrate relatively high reduction potentials for these complexes, leading to the formation of Ru(II) species in aqueous solutions containing biological reducing agents, such as ascorbate. However, EPR measurements indicate that while noncovalent interactions with hsA do not prevent reduction, covalent binding produces persistent mononuclear Ru(III) species under these conditions.

This report describes the reduction of a niobium(V) phthalocyanine complex and investigation of the electronic structure of the resulting products. The reduction of PcNbCl(3) (Pc = phthalocyanine dianion) with 5.5 equiv of potassium graphite in 1,2-dimethoxyethane (DME) resulted in the isolation of K(2)PcNbO.5DME (1a). Addition of 18-crown-6 to 1a gave [K(18-crown-6)](2)(mu-DME)PcNbO (1b). Both 1a and 1b were structurally characterized by single-crystal X-ray diffraction analysis. In both complexes, the niobium center adopts a square pyramidal geometry and is coordinated by four basal Pc nitrogen atoms and an apical oxo ligand. Notably, the Pc ligand in 1a is saddle-shaped, with significant bond length alternation, rather than flat with delocalized bonding. The production of ethylene during the reduction of PcNbCl(3), detected by gas chromatography/mass spectrometry (GC/MS), suggests that the oxo ligand likely results from double C-O bond activation of DME solvent. A combination of spectroscopic techniques and density functional theory (DFT) calculations were used to establish the electronic structure of 1a. The close correspondence of the electronic absorption spectrum of 1a to that of [PcZn](2-) with a di-reduced Pc(4-) ligand, indicates a similar electronic structure for the two complexes. Evaluation of the electronic transitions for 1a and [PcZn](2-) by time-dependent DFT calculations further suggests a similar electronic structure for both complexes, indicating that differences in symmetry between 1a and [PcZn](2-) do not significantly affect the nature of the electronic transitions. Electron paramagnetic resonance (EPR) spectroscopy of 1a in solution at room temperature gave a 10-line spectrum, while frozen-solution X- and Q-band EPR spectra are consistent with powder-pattern spectra defined by uniaxial g and (93)Nb hyperfine tensors: these imply the presence of a d(1) Nb(IV) metal center. EPR and electron nuclear double resonance spectroscopy suggests that the spin density in 1a is centered almost completely on the niobium, in agreement with the DFT calculations. These results illustrate the value of Pc as a chemically inert, redox-active ligand for stabilizing reactive metal centers.

Phosphonium ionic liquids (PhosILs), most notably tetradecyl(trihexyl)phosphonium decanoate (PhosIL-C(9)H(1)9COO), are solvents for bases such as Grignard reagents, isocyanides, Wittig reagents (phosphoranes), and N-heterocyclic carbenes (NHCs). The stability of the organometallic species in PhosIL solution is anion dependent. Small bases, such as hydroxide, react with the phosphonium ions and promote C-H exchange as suggested by deuterium-labeling studies. A method to dry and purify the ionic liquids is described and this step is important for the successful use of basic reagents in PhosIL. NHCs have been generated in PhosIL, and these persistent solutions catalyze organic transformations such as the benzoin condensation and the Kumada-Corriu cross-coupling reaction. Phosphoranes were generated in PhosIL, and their reactivity with various organic reagents was also tested. Inter-ion contacts involving tetraalkylphosphonium ions have been assessed, and the crystal structure of [(n-C(4)H(90)(4)P][CH(3)CO(2).CH(3)CO(2)H] has been determined to aid the discussion. Decomposition of organometallic compounds may also proceed through electron-transfer processes that, inter alia, may lead to decomposition of the IL, and hence the electrochemistry of some representative phosphonium and imidazolium ions has been studied. A radical derived from the electrochemical reduction of an imidazolium ion has been characterized by electron paramagnetic resonance spectroscopy.

The bulky m-terphenyl phenol Ar*OH 1 (Ar* = C6H3-2,6-Mes2, Mes = 2,4,6-trimethylphenyl) was synthesized via the treatment of Ar*Li with nitrobenzene. The phenol 1 is prepared in modest to good yield using this method. Attempts were also made to prepare 1 through oxidation of the bulky boronic acid Ar*B(OH)2with Oxone®, but this reaction was not suitable for preparative-scale reactions. Side products of the reaction between Ar*Li and nitrobenzene were identified as Ar*[N(O)Ph] and [C6H5N(O)]2and were characterized by X-ray crystallography and EPR spectroscopy. A variety of main-group and transition-metal complexes of Ar*OH were prepared, namely Sn(OAr*)2, Ge(OAr*)2, [N(SiMe3)2]Ge(OAr*), [Me2Al(OAr*)]2, and Ti(NMe2)(OAr*)2. All compounds were characterized spectroscopically and most were studied by single-crystal X-ray diffraction as well.Key words: m-terphenyl, main-group compounds, X-ray crystallography, multinuclear NMR spectroscopy, EPR spectroscopy.

Pummerer-like rearrangements of carbohydrate-based heterocycles containing selenium and sulfur were investigated. To the best of our knowledge, this is the first report on the Pummerer rearrangement in selenoheterocycles. Ozonization of 1,4-anhydro-D-galactitol or 1,5-anhydroxylitol derivatives containing sulfur or selenium as the ring heteroatom gave unstable intermediates that were attributed to ozonides. These intermediates decomposed upon warming to give selenoxides or sulfoxides. Significantly, addition of acetic anhydride at low temperature to the ozonization reaction mixtures gave Pummerer-rearrangement products after warming to ambient temperature. However, when the isolated selenoxides or sulfoxides were treated with acetic anhydride, Pummerer rearrangement occurred but the sulfoxides required much higher reaction temperatures. The latter results are at variance with the former and are interpreted in terms of the rearrangement of the ozonide acetate intermediates in the former cases. To probe whether the rearrangement proceeded heterolytically via extrusion of singlet oxygen or homolytically via the generation of radical species, trapping experiments with rubrene and electron paramagnetic resonance (EPR) studies with the radical trap DMPO were performed. The results of these experiments are consistent with the intermediacy of radical species and suggest a new and milder synthetic method to generate Pummerer-type products.

This article outlines the present state of knowledge of paramagnetic defects in crystalline zircon as obtained mainly, but not exclusively, from electron paramagnetic resonance (EPR) studies in crystalline zircon (zirconium silicate, ZrSiO4). The emphasis is on single-crystal studies where, in principle, unambiguous analysis is possible. Firstly, the crystallography of zircon is presented. Secondly, the relationships between available crystal-site symmetries and the symmetries of observed paramagnetic species in zircon, and how these observations lead to unambiguous assignments of point-group symmetries for particular paramagnetic species are detailed. Next, spin-Hamiltonian (SH) analysis is discussed with emphasis on the symmetry relationships that necessarily exist amongst the Laue classes of the crystal sites in zircon, the paramagnetic species occupying those sites and the SH itself. The final sections of the article then survey the results of EPR studies on zircon over the period 1960–2002.

X-band electron paramagnetic resonance experiments on a gamma-irradiated α-quartz crystal have revealed a new hydrogarnet-like defect. Analysis of the spectral data suggests that the centre consists of an oxygenic electron hole adjacent to a silicon vacancy, charge-compensated by a proton and two lithium +1 ions. The principal directions of the spin-Hamiltonian g matrix indicate that the oxygenic hole occurs on the oxygen ion that would be long-bonded to the missing silicon atom. Modelling of the proton and lithium hyperfine matrices via three different methods has provided a detailed physical view for the structure of the defect centre, which is presented here.

A new study, by electron paramagnetic resonance (EPR) spectroscopy at cryogenic temperatures, of aluminum oxygenic-hole centres in a α-quartz single crystal is reported. The well-known centre [AlO4]0 has been reinvestigated with somewhat improved techniques and computer analysis, confirming previous results in the literature. A previously unreported Al–Li centre has now been well characterized by EPR. From the fitted parameter matrices g, A(27Al), P(27Al), A(7Li), and P(7Li) we have determined not only that the hole resides on a "long-bonded" oxygen anion, as in [AlO4]0, but have also established the position of the near-by interstitial lithium ion. One model for this centre is that it consists of a lithium atom Li0 linked to the diamagnetic centre [AlO4]–, yielding hole-bearing species [AlO4/Li]– via internal charge transfer. PACS Nos.: 7630Da, 7630Mi

Alzheimer's disease (AD) is a neurodegenerative disorder that is increasing worldwide due to increased life expectancy. AD is characterized by two pathological hallmarks in the brain: amyloid-β (Aβ) plaque deposits and neurofibrillary tangles. A focus of AD research has concentrated on either inhibiting Aβ peptide aggregation that leads to plaque formation or breaking down pre-formed Aβ peptide aggregates. An alternative approach is to modulate the Aβ aggregation profile by facilitating the formation of Aβ species that are off-pathway and non-toxic. Herein, we report the re-purposing of the widely studied Ru(III) anti-cancer complex KP1019, towards regulating the aggregation profile of the Aβ peptide. Using electron paramagnetic resonance (EPR) spectroscopy, we conclude that KP1019 binds to histidine residues, located at the N-terminus of the peptide, in a rapid and robust fashion. Native gels and transmission electron microscopy (TEM) analyses have provided insight into the species and structures that are generated by KP1019-Aβ interactions. Finally, incubation in an in vitro human neuronal cell model has demonstrated that the formation of KP1019-Aβ species rescues cell viability from Aβ-associated neurotoxicity. Modulation of the Aβ aggregation pathway via covalent interactions with small molecules is thus a promising AD therapeutic strategy.

Anthrax lethal factor (LF) is a zinc-dependent metalloendopeptidase and a member of the gluzincin family. The current report demonstrates a high metal substitution tolerance of LF atypical of gluzincins and other zinc-dependent metalloproteases. Mn(2+), Co(2+), Ni(2+), Cu(2+) and Cd(2+) were found to reactivate the apoprotein of LF to a level either comparable to or significantly higher than that noted for the native zinc enzyme. The most active form of LF was obtained with Cu(2+), a surprising observation since most Cu(2+)-substituted zinc proteases display very low activity. Cu(2+)-substituted LF (CuLF), prepared by direct exchange and by apoprotein reconstitution methodologies, displayed a several-fold higher catalytic competence towards chromogenic and fluorogenic LF substrates than native LF. CuLF bound Cu(2+) tightly with a dissociation constant in the femtomolar range. The electron paramagnetic resonance spectrum of CuLF revealed the protein-bound metal ion to be coordinated to two nitrogen donor atoms, suggesting that Cu(2+) binds to both active site histidine residues. While ZnLF and CuLF (prepared by direct exchange) were capable of killing RAW 264.7 murine macrophage-like cells, apoLF and all metal-reconstituted apoprotein preparations failed to elicit a cytotoxic response. Competition experiments using apoLF/ZnLF mixtures demonstrated the propensity of apoLF to relieve ZnLF-induced cell death, suggesting that both protein forms can compete with each other for binding to protective antigen. The lack of cytotoxicity of apoLF and its metal-reconstituted variants likely originates from structural perturbations in these proteins which might prevent their translocation into the cytoplasm.

We introduce a new simple methodology allowing the measurement of (1)H-(15)N residual dipolar couplings, dipolar shifts, and unpaired electron-amide proton distances. This method utilizes a zinc finger tag fused at either the N- or the C-terminus of a protein. We have demonstrated this fusion strategy by incorporating the zinc finger of the retroviral gag protein onto the C-terminus of barnase, a ribonuclease produced by Bacillus amiloliquifaciance. We show that this tag can be substituted with cobalt and manganese. Binding of cobalt to the gag zinc finger-barnase fusion protein introduced sufficient anisotropic paramagnetic susceptibility for orientation of the molecule in the magnetic field. Partial alignment permitted measurement of (1)J(HN) scalar couplings along with dipolar couplings. Replacement of bound cobalt with diamagnetic zinc removes the paramagnetic-induced orientation of barnase, permitting the measurement of only (1)J(HN) scalar couplings. Dipolar couplings, ranging from -0.9 to 0.6 Hz, were easily measured from the difference in splitting frequencies in the presence of cobalt and zinc. The observed paramagnetic anisotropy induced by cobalt binding to the metal binding tag also permitted measurement of dipolar shifts. Substitution of manganese into the metal binding tag permitted the measurement of unpaired electron-amide proton distances using paramagnetic relaxation enhancement methodology. The availability of both amide proton dipolar shifts and unpaired electron to amide proton distances permitted the direct calculation of z-coordinates for individual amide protons. This approach is robust and will prove powerful for global fold determination of proteins identified in genome initiatives.

The Zr(α) centre is the most prominent centre observed after low temperature x-irradiation of zirconium silicate (zircon). The centre has been identified as due to an electron trap in the form of a Zr3+ ion in the zircon lattice with the centre being stabilized by interaction with a 100% abundant I = 1/2 ion. Previous reports have discussed the Zr(α) centre in terms of an 89 Y3+ (I = 1/2) ion in a neighbouring Zr4+ site. Using both EPR and ENDOR it has now been possible to show unequivocally that the I = 1/2 nuclear interaction is due to the presence of a 31P ion. Detailed analysis of the g and hyperfine matrices for 91Zr, 31P and 29Si is used to develop a model of the centre as an electron trapped on zirconium as a Zr3+ ion with a P5+ ion in the nearest Si4+ site on the mirror plane. The defect is now best described as a [ZrPO4]0 centre.

Precise interaction matrices are reported for a boron-stabilized oxygenic-hole centre in x-irradiated zircon as derived from x-band electron paramagnetic resonance (EPR) measurements at ca 15 K. The presence of boron has been identified unequivocally through the observation and spin Hamiltonian analysis of superhyperfine structures from both naturally occurring isotopes of boron. Each of the parameter matrices indicates that the centre has point-group symmetry m (Cs ), as is common in hole centres in zircon, where the hole centre is trapped on an oxygen atom within one of the crystal mirror planes. Analysis of the principal directions of these matrices indicates that the boron atom resides in a silicon position neighbouring the oxygenic electron vacancy.

A surprisingly large number of paramagnetic hole centres in zircon have been observed and characterized by EPR. The field is reviewed and a model proposed based on some ten precisely measured centres. EPR data suggest that the trapped hole, produced by x-irradiation, is normally located in the non-bonding pz orbital of an O- ion and is stabilized by a nearby impurity cation, often, but not always, a +3 ion. Two previously unobserved oxygenic-hole centres are here reported. In one the stabilizing ion is shown from observed 89 Y superhyperfine structure to be Y3+ and conforms to the above model. The second has the most anisotropic g values yet observed in an oxygenic-hole centre in zircon. Two g values are significantly below the free-electron value and a px ground state is inferred.

Two previously unreported Ti3+ paramagnetic centres have been observed in zircon (zirconium silicate, ZrSiO4) and studied by X-band electron paramagnetic resonance at 10 K. The first of these is shown to arise from a Ti3+ electron-trap centre where Ti substitutes into one of the Si4+ sites, point group symmetry 2m (D2d), of the tetragonal crystal. A full spin-Hamiltonian analysis of the centre is given. The second centre arises from Ti3+ substituting for Zr4+ in the lattice and interacting with a 100% spin-I = ½ nucleus, probably 31P, in a nearby lattice site, thought to be a next-nearest-neighbour Si site. The point group symmetry of the site of this centre is 2 (C2).

A new centre formed by x-irradiation of a synthetic zircon crystal at 77 K has been found and studied by electron paramagnetic resonance at 10 K. The impurity ion is believed to occupy a substitutional site, as was the case for the earlier reported B centre. Interaction with an nucleus in a nearest-neighbour site lowers the site symmetry from (Laue class, 4/mmm) tetragonal to m (Laue class, 2/m) monoclinic.

CX-5461 is a RNA polymerase I inhibitor currently in Phase I clinical trial in Australia for patients with advanced hematologic malignancies. In the pre-clinical setting, CX-5461 is efficacious in a wide range of hematologic and solid tumor models when given orally or intraperitoneally. Currently, the compound is given intravenously (iv) in the first-in-human clinical trial. CX-5461 is solubilized in low pH (3.5) 50 mM sodium phosphate as it is sparingly soluble in water ( Citation Format: Ada W.Y. Leung, Malathi Anantha, Kathleen E. Prosser, Mohamed Wehbe, Charles J. Walsby, Marcel B. Bally. A novel formulation of CX-5461, a small-molecule inhibitor of rRNA synthesis, and its use for treatment of acute myeloid leukemia models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1319.

In this paper we demonstrate that the Kubas interaction, a nondissociative form of weak hydrogen chemisorption with binding enthalpies in the ideal 20-30 kJ/mol range for room-temperature hydrogen storage, can be exploited in the design of a new class of hydrogen storage materials which avoid the shortcomings of hydrides and physisorpion materials. This was accomplished through the synthesis of novel vanadium hydrazide gels that use low-coordinate V centers as the principal Kubas H(2) binding sites with only a negligible contribution from physisorption. Materials were synthesized at vanadium-to-hydrazine ratios of 4:3, 1:1, 1:1.5, and 1:2 and characterized by X-ray powder diffraction, X-ray photoelectron spectroscopy, nitrogen adsorption, elemental analysis, infrared spectroscopy, and electron paramagnetic resonance spectroscopy. The material with the highest capacity possesses an excess reversible storage of 4.04 wt % at 77 K and 85 bar, corresponding to a true volumetric adsorption of 80 kg H(2)/m(3) and an excess volumetric adsorption of 60.01 kg/m(3). These values are in the range of the ultimate U.S. Department of Energy goal for volumetric density (70 kg/m(3)) as well as the best physisorption material studied to date (49 kg H(2)/m(3) for MOF-177). This material also displays a surprisingly high volumetric density of 23.2 kg H(2)/m(3) at room temperature and 85 bar--roughly 3 times higher than that of compressed gas and approaching the DOE 2010 goal of 28 kg H(2)/m(3). These materials possess linear isotherms and enthalpies that rise on coverage and have little or no kinetic barrier to adsorption or desorption. In a practical system these materials would use pressure instead of temperature as a toggle and can thus be used in compressed gas tanks, currently employed in many hydrogen test vehicles, to dramatically increase the amount of hydrogen stored and therefore the range of any vehicle.

The compounds imidazolium [trans-[RuCl(4)(1H-imidazole)(2)] (KP418) and indazolium [trans-RuCl(4)(1H-indazole)(2)] (KP1019) both show significant anticancer activity, with the latter recently having completed phase I clinical trials. An important component of this success has been associated with targeted delivery of the complexes to cancer cells by serum proteins. In this study, electron paramagnetic resonance (EPR) measurements, combined with incubation under physiological conditions, and separation of protein-bound fractions, have been used to characterize the interactions of these complexes with human serum albumin (hsA), human serum transferrin (hsTf) apoprotein, and whole human serum. The strong EPR signals observed in these experiments demonstrate that both complexes are primarily retained in the 3+ oxidation state in the presence of serum components. Rapid, noncovalent binding of KP1019 was observed in the presence of both hsA and serum, indicating that the predominant interactions occur within the hydrophobic binding sites of hsA. This sequestering process correlates with the low levels of side effects observed in clinical trials of the complex. At longer incubation times, the noncovalently bound complexes are converted slowly to a protein-coordinated form. Noncovalent interactions are not observed in the presence apo-hsTf, where only slow binding of KP1019 via ligand exchange with the protein occurs. By contrast, hydrophobic interactions of KP418 with hsA only occur with the aquated products of the complex, a process that also dominates in serum. In the presence of apo-hsTf, KP418 interacts directly with the protein through exchange of ligands, as observed with KP1019.

Grignard reagents form persistent solutions in phosphonium ionic liquids possessing O-donor anions and these solutions are excellent reaction media for electron transfer processes and transmetallation reactions.

Lysine 2,3-aminomutase (LAM) utilizes a [4Fe-4S] cluster, S-adenosyl-L-methionine (SAM), and pyridoxal 5'-phosphate (PLP) to isomerize L-alpha-lysine to L-beta-lysine. LAM is a member of the radical-SAM enzyme superfamily in which a [4Fe-4S]+ cluster reductively cleaves SAM to produce the 5'-deoxyadenosyl radical, which abstracts an H-atom from substrate to form 5'-deoxyadenosine (5'-Ado) and the alpha-Lys* radical (state 3 (Lys*)). This radical isomerizes to the beta-Lys* radical (state 4(Lys*)), which then abstracts an H-atom from 5'-Ado to form beta-lysine and the 5'-deoxyadenosyl radical; the latter then regenerates SAM. We use 13C, 1,2H, 31P, and 14N ENDOR to characterize the active site of LAM in intermediate states that contain the isomeric substrate radicals or analogues. With L-alpha-lysine as substrate, we monitor the state with beta-Lys*. In parallel, we use two substrate analogues that generate stable analogues of the alpha-Lys* radical: trans-4,5-dehydro-L-lysine (DHLys) and 4-thia-L-lysine (SLys). This first glimpse of the motions of active-site components during catalytic turnover suggests a possible major movement of PLP during catalysis. However, the principal focus of this work is on the relative positions of the carbons involved in H-atom transfer. We conclude that the active site facilitates hydrogen atom transfer by enforcing van der Waals contact between radicals and their reacting partners. This constraint enables the enzyme to minimize and even eliminate side reactions of highly reactive species such as the 5'-deoxyadensosyl radical.

FosA is a manganese metalloglutathione transferase that confers resistance to the broad-spectrum antibiotic fosfomycin, which contains a phosphonate group. The active site of this enzyme consists of a high-spin Mn(2+) ion coordinated by endogenous ligands (a glutamate and two histidine residues) and by exogenous ligands, such as substrate fosfomycin. To study the Mn(2+) coordination environment of FosA in the presence of substrate and the inhibitors phosphonoformate and phosphate, we have used (31)P pulsed electron-nuclear double resonance (ENDOR) at 35 GHz to obtain metrical information from (31)P-Mn(2+) interactions. We have found that continuous wave (CW) (31)P ENDOR is not successful in the study of phosphates and phosphonates coordinated to Mn(2+). Parallel studies of phosph(on)ate binding to the Mn(2+) of FosA and to aqueous Mn(2+) ion disclose how the enzyme modifies the coordination of these molecules to the active site Mn(2+). Through analysis of (31)P hyperfine parameters derived from simulations of the ENDOR spectra we have determined the binding modes of the phosph(on)ates in each sample and discerned details of the geometric and electronic structure of the metal center. The (31)P ENDOR studies of the protein samples agree with, or improve on, the Mn-P distances determined from crystal structures and provide Mn-phosph(on)ate bonding information not available from these studies. Electron spin echo electron paramagnetic resonance (ESE-EPR) spectra have also been recorded. Simulation of these spectra yield the axial and rhombic components of the Mn(2+) (S = (5)/(2)) zero-field splitting (zfs) tensor. Comparison of structural inferences based on these zfs parameters both with the known enzyme structures and the (31)P ENDOR results establishes that the time-honored procedure of analyzing Mn(2+) zfs parameters to describe the coordination environment of the metal ion is not valid or productive.

Electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopies and other physical methods have provided important new insights into the radical-SAM superfamily of proteins, which use iron-sulfur clusters and S-adenosylmethionine to initiate H atom abstraction reactions. This remarkable chemistry involves the generation of the extremely reactive 5'-deoxyadenosyl radical, the same radical intermediate utilized in B12-dependent reactions. Although early speculation focused on the possibility of an organometallic intermediate in radical-SAM reactions, current evidence points to novel chemistry involving a site-differentiated [4Fe-4S] cluster. The focus of this forum article is on one member of the radical-SAM superfamily, pyruvate formate-lyase activating enzyme, and how physical methods, primarily EPR and ENDOR spectroscopies, are contributing to our understanding of its structure and mechanism. New ENDOR data supporting coordination of the methionine moiety of SAM to the unique site of the [4Fe-4S]2+/+ cluster are presented.