Directed Evolution of a Cp*RhIII -Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification.

Directed evolution of Cp*RhIII -linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII -linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.

Structural and Photodynamic Studies on Nitrosylruthenium-Complexed Serum Albumin as a Delivery System for Controlled Nitric Oxide Release.

How to deliver nitric oxide (NO) to a physiological target and control its release quantitatively is a key issue for biomedical applications. Here, a water-soluble nitrosylruthenium complex, [(CH3)4N][RuCl3(5cqn)(NO)] (H5cqn = 5-chloro-8-quinoline), was synthesized, and its structure was confirmed with 1H NMR and X-ray crystal diffraction. Photoinduced NO release was investigated with time-resolved Fourier transform infrared and electron paramagnetic resonance (EPR) spectroscopies. The binding constant of the [RuCl3(5cqn)(NO)]- complex with human serum albumin (HSA) was determined by fluorescence spectroscopy, and the binding mode was identified by X-ray crystallography of the HSA and Ru-NO complex adduct. The crystal structure reveals that two molecules of the Ru-NO complex are located in the subdomain IB, which is one of the major drug binding regions of HSA. The chemical structures of the Ru complexes were [RuCl3(5cqn)(NO)]- and [RuCl3(Glycerin)NO]-, in which the electron densities for all ligands to Ru are unambiguously identified. EPR spin-trapping data showed that photoirradiation triggered NO radical generation from the HSA complex adduct. Moreover, the near-infrared image of exogenous NO from the nitrosylruthenium complex in living cells was observed using a NO-selective fluorescent probe. This study provides a strategy to design an appropriate delivery system to transport NO and metallodrugs in vivo for potential applications.

Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter.

The calcium (Ca2+) uniporter of mitochondria is a holocomplex consisting of the Ca2+-conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca2+ selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn2+), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn2+ specifically, whereas in the WT the two rings bind Mn2+ cooperatively, resulting in ∼1,000-fold higher apparent affinity. Ca2+ can specifically displace the bound Mn2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn2+ binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.

DNA Binding and Photocleavage Properties, Cellular Uptake and Localization, and in-Vitro Cytotoxicity of Dinuclear Ruthenium(II) Complexes with Varying Lengths in Bridging Alkyl Linkers.

Two new dinuclear Ru(II) polypyridyl complexes containing three and ten methylene chains in their bridging linkers are synthesized and characterized. Their calf thymus DNA-binding and plasmid DNA photocleavage behaviors are comparatively studied with a previously reported, six-methylene-containing analog by absorption and luminescence spectroscopy, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, DNA viscosity measurements, DNA thermal denaturation, and agarose gel electrophoresis analyses. Theoretical calculations applying the density functional theory (DFT) method for the three complexes are also performed to understand experimentally observed DNA binding properties. The results show that the two complexes partially intercalate between the base pairs of DNA. Cellular uptake and colocalization studies have demonstrated that the complexes could enter HeLa cells efficiently and localize within lysosomes. The in-vitro antitumor activity against HeLa and MCF-7 tumor cells of the complexes are studied by MTT cytotoxic analysis. A new method, high-content analysis (HCA), is also used to assess cytotoxicity, apoptosis and cell cycle arrest of the three complexes. The results show that the lengths of the alkyl linkers could effectively tune their biological properties and that HCA is suitable for rapidly identifying cytotoxicity and can be substituted for MTT assays to evaluate the cell cytotoxicity of chemotherapeutic agents.

Dinuclear polypyridylruthenium(II) complexes: flow cytometry studies of their accumulation in bacteria and the effect on the bacterial membrane.

OBJECTIVES: To determine the energy dependency of and the contribution of the membrane potential to the cellular accumulation of the dinuclear complexes [{Ru(phen)2}2{µ-bbn}](4+) (Rubbn) and the mononuclear complexes [Ru(Me4phen)3](2+) and [Ru(phen)2(bb7)](2+) in Staphylococcus aureus and Escherichia coli, and to examine their effect on the bacterial membrane. METHODS: The accumulation of the ruthenium complexes in bacteria was determined using flow cytometry at a range of temperatures. The cellular accumulation of the ruthenium complexes was also determined in cells that had been incubated with the metal complexes in the presence or absence of metabolic stimulators or inhibitors and/or commercial dyes to determine the membrane potential or membrane permeability. RESULTS: The accumulation of ruthenium complexes in the two bacterial strains was shown to increase with increasing incubation temperature, with the relative increase in accumulation greater with E. coli, particularly for Rubb12 and Rubb16. No decrease in accumulation was observed for Rubb12 in ATP-inhibited cells. While carbonyl cyanide m-chlorophenyl hydrazone (CCCP) did depolarize the cell membrane, no reduction in the accumulation of Rubb12 was observed; however, all ruthenium complexes, when incubated with S. aureus at concentrations twice their MIC, depolarized the membrane to a similar extent to CCCP. Except for the mononuclear complex [Ru(Me4phen)3](2+), incubation of any of the other ruthenium complexes allowed a greater quantity of the membrane-impermeable dye TO-PRO-3 to be taken up by S. aureus. CONCLUSIONS: The results indicate that the potential new antimicrobial Rubbn complexes enter the cell in an energy-independent manner, depolarize the cell membrane and significantly permeabilize the cellular membrane.

Long-lasting hypotensive effect in renal hypertensive rats induced by nitric oxide released from a ruthenium complex.

In this study, we investigated the effect of the ruthenium complex [Ru(terpy)(bdq)NO] (TERPY) on the arterial pressure from renal hypertensive 2 kidney-1 clip (2K-1C) rats, which was compared with sodium nitroprusside (SNP). The most interesting finding was that the intravenous bolus injection of TERPY (2.5, 5.0, 7 mg/kg) had a dose-dependent hypotensive effect only in 2K-1C rats. On the other hand, SNP (35 and 70 µg/kg) presented a similar hypotensive effect in both normotensive (2K) and 2K-1C although the effect of 70 µg/kg was >35 µg/kg. The injection of the nonselective NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) increased the arterial pressure in 2K and 2K-1C rats with a similar magnitude. After infusion of L-NAME, the hypotensive effect induced by TERPY and SNP was potentiated in both 2K and in 2K-1C rats. The administration of the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl increased the hypotensive effect induced by TERPY or SNP in both 2K and 2K-1C rats. The hypotensive effect induced by TERPY was longer than that produced by SNP. Taken together, our results show that the TERPY has a long-lasting hypotensive effect, which has a dose dependence and higher magnitude in 2K-1C compared with in 2K rats. In comparison with SNP, TERPY is less potent in inducing arterial pressure fall, but it presents a much longer hypotensive effect.

Biomolecular Interactions of Cytotoxic Ruthenium Compounds with Thiosemicarbazone or Benzothiazole Schiff Base Chelates.

Herein we illustrate the formation and characterization of new paramagnetic ruthenium compounds, trans-P-[RuCl(PPh3 )2 (pmt)]Cl (1) (Hpmt=1-((pyridin-2-yl)methylene)thiosemicarbazide), trans-P-[RuCl(PPh3 )2 (tmc)]Cl (2) (Htmc=1-((thiophen-2-yl)methylene)thiosemicarbazide) and a diamagnetic ruthenium complex, cis-Cl, trans-P-[RuCl2 (PPh3 )2 (btm)] (3) (btm=2-((5-hydroxypentylimino)methyl)benzothiazole). Agarose gel electrophoresis experiments of the metal compounds illustrated dose-dependent binding to gDNA by 1-3, while methylene blue competition assays suggested that 1 and 2 are also DNA intercalators. Assessment of the effects of the compounds on topoisomerase function indicated that 1-3 are capable of inhibiting topoisomerase I activity in terms of the ability to nick supercoiled plasmid DNA. The cytotoxic activities of the metal complexes were determined against a range of cancer cell lines versus a non-tumorigenic control cell line, and the complexes were, in general, more cytotoxic towards the cancer cells, displaying IC50 values in the low micromolar range. Time-dependent stability studies showed that in the presence of strong nucleophilic species (such as DMSO), the chloride co-ligands of 1-3 are rapidly substituted by the former as proven by the suppression of the substitution reactions in the presence of an excess amount of chloride ions. The metal complexes are significantly stable in both DCM and an aqueous phosphate buffer containing 2 % DMSO.

Ruthenium-nitrite complex as pro-drug releases NO in a tissue and enzyme-dependent way.

Nitric oxide (NO) plays an important role in the control of the vascular tone and the most often employed NO donors have limitations due to their harmful side-effects. In this context, new NO donors have been prepared, in order to minimize such undesirable effects. cis-[Ru(bpy)2(py)NO2](PF6) (RuBPY) is a new nitrite complex synthesized in our laboratory that releases NO in the presence of the vascular tissue only. In this work the vasorelaxation induced by this NO donor has been studied and compared to that obtained with the well known NO donor SNP. The relaxation induced by RuBPY is concentration-dependent in denuded rat aortas pre-contracted with phenylephrine (EC50). This new compound induced relaxation with efficacy similar to that of SNP, although its potency is lower. The time elapsed until maximum relaxation is achieved (E max=240s) is similar to measured for SNP (210s). Vascular reactivity experiments demonstrated that aortic relaxation by RuBPY is inhibited by the soluble guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiozolo[4,3-a]quinoxaline-1-one (ODQ 1µM). In a similar way, 1µM ODQ also reduces NO release from the complex as measured with DAF-2 DA by confocal microscopy. These findings suggest that this new complex RuBPY that has nitrite in its structure releases NO inside the vascular smooth muscle cell. This ruthenium complex releases significant amounts of NO only in the presence of the aortic tissue. Reduction of nitrite to NO is most probably dependent on the soluble guanylyl-cyclase enzyme, since NO release is inhibited by ODQ.

Model study of CO inhibition of [NiFe]hydrogenase.

We propose a modified mechanism for the inhibition of [NiFe]hydrogenase ([NiFe]H(2)ase) by CO. We present a model study, using a NiRu H(2)ase mimic, that demonstrates that (i) CO completely inhibits the catalytic cycle of the model compound, (ii) CO prefers to coordinate to the Ru(II) center rather than taking an axial position on the Ni(II) center, and (iii) CO is unable to displace a hydrido ligand from the NiRu center. We combine these studies with a reevaluation of previous studies to propose that, under normal circumstances, CO inhibits [NiFe]H(2)ase by complexing to the Fe(II) center.

Redox, spectroscopic, and photophysical properties of Ru-Pt mixed-metal complexes incorporating 4,7-diphenyl-1,10-phenanthroline as efficient DNA binding and photocleaving agents.

The redox, spectroscopic, and photophysical properties as well as DNA interactions of the new bimetallic complexes [(Ph2phen)2Ru(BL)PtCl2](2+) (Ph2phen = 4,7-diphenyl-1,10-phenanthroline, and BL (bridging ligand) = dpp = 2,3-bis(2-pyridyl)pyrazine, or dpq = 2,3-bis(2-pyridyl)quinoxaline) were investigated. These Ru-polyazine chromophores with Ph2phen TLs (terminal ligands) and polyazine BLs are efficient light absorbers. The [(Ph2phen)2Ru(BL)PtCl2](2+) complexes display reversible Ru(II/III) oxidations at 1.57 (dpp) and 1.58 (dpq) V vs SCE (saturated calomel electrode) with an irreversible Pt(II/IV) oxidation occurring prior at 1.47 V vs SCE. Four, reversible ligand reductions occur at -0.50 dpp(0/-), -1.06 dpp(-/2-), -1.37 Ph2phen(0/-), and -1.56 V vs SCE Ph2phen(0/-). For the [(Ph2phen)2Ru(dpq)PtCl2](2+) complex, the first two reductions shift to more positive potentials at -0.23 and -0.96 V vs SCE. The electronic absorption spectroscopy is dominated in the UV region by π → π* ligand transitions and in the visible region by metal-to-ligand charge transfer (MLCT) transitions at 517 nm for [(Ph2phen)2Ru(dpp)PtCl2](2+) and 600 nm for [(Ph2phen)2Ru(dpq)PtCl2](2+). Emission spectroscopy shows that upon attaching Pt to the Ru monometallic precursor the λmax(em) shifts from 664 nm for [(Ph2phen)2Ru(dpp)](2+) to 740 nm for [(Ph2phen)2Ru(dpp)PtCl2](2+). The cis-Pt(II)Cl2 bioactive site offers the potential of targeting DNA by covalently binding the mixed-metal complex to DNA bases. The multifunctional interactions with DNA were assayed using both linear and circular plasmid pUC18 DNA gel shift assays. Both title complexes can bind to and photocleave DNA with dramatically enhanced efficiency relative to previously reported systems. The impact of the Ph2phen TL on photophysics and bioreactivity is somewhat surprising given the Ru → BL charge transfer (CT) nature of the photoreactive state in the complexes.

Red-Light-Responsive Ru Complex Photosensitizer for Lysosome Localization Photodynamic Therapy.

Photoresponsive ruthenium (Ru) complexes have been extensively studied in the photodynamic therapy (PDT) of cancer. The metal-to-ligand charge transfer (MLCT) absorption maximum of most Ru complexes is located in the short-wavelength visible region, which is well suited for superficial tumors but shows inefficient therapeutic effects for more deep-seated ones. Moreover, Ru complexes are primarily located in the mitochondria or nucleus, always resulting in high levels of dark toxicity and DNA mutation. Herein, we reported a new ruthenium complex (Ru-I) for red-light-triggered PDT. The activation wavelength of Ru-I is successfully extended to 660 nm. Importantly, the complex photosensitizer can be quickly taken up by cancer cells and selectively accumulated in the lysosome, an ideal localization for PDT purposes. Intratumoral injection of Ru-I into tumor-bearing mice achieved excellent therapeutic effects and thus holds great promise for applications in lysosome localization photodynamic therapy.

Strained, Photoejecting Ru(II) Complexes that are Cytotoxic Under Hypoxic Conditions.

A series of strained Ru(II) complexes were studied for potential anticancer activity in hypoxic tissues. The complexes were constructed with methylated ligands that were photolabile and an imidizo[4,5-f][1,10]phenanthroline ligand that contained an appended aromatic group to potentially allow for contributions of ligand-centered excited states. A systematic variation of the size and energy of the aromatic group was performed using systems containing 1-4 fused rings, and the photochemical and photobiological behaviors of all complexes were assessed. The structure and nature of the aromatic group had a subtle impact on photochemistry, altering environmental sensitivity, and had a significant impact on cellular cytotoxicity and photobiology. Up to 5-fold differences in cytotoxicity were observed in the absence of light activation; this rose to 50-fold differences upon exposure to 453 nm light. Most significantly, one complex retained activity under conditions with 1% O2 , which is used to induce hypoxic changes. This system exhibited a photocytotoxicity index (PI) of 15, which is in marked contrast to most other Ru(II) complexes, including those designed for O2 -independent mechanisms of action.

Ruthenium(II) complexes of bipyridine-glycoluril and their interactions with DNA.

Nine complexes of the type [Ru(N-N)(2)(BPG)]Cl(2) 1-4, [Ru(N-N)(BPG)(2)]Cl(2) 5-8, and [Ru(BPG)(3)]Cl(2) 9 where N-N is 2,2'-bipyridine (bpy), 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz), which incorporates bipyridine-glycoluril (BPG-4b,5,7,7a-tetrahydro-4b,7a-epiminomethanoimino-6H-imidazo[4,5-f][1,10]phenanthroline-6,13-dione) as the ancillary ligand, have been synthesized and characterized. These complexes with the peripheral polypyridyl ligands have the ability to form conjugates with DNA. The DNA binding (absorption spectroscopy, steady-state and time-resolved emission measurements, steady-state emission quenching measurements) and cleavage (under dark and irradiated conditions) by these complexes has been studied to investigate the influence of the ancillary ligand. The binding ability of these complexes to DNA is dependent on the planarity of the intercalative polypyridyl ligand, which is further affected by the ancillary bipyridine-glycoluril ligand. The complexes 3, 4, 7, and 8 bind to CT-DNA with binding constants on the order of 10(4) M(-1). Time-resolved emission measurements on the DNA-bound complexes 1, 3, 5-7, and 9 show monoexponential decay of the excited states, whereas complexes 2, 4, and 8 show biexponential decay with short- and long-lived components. Interaction of complexes 2-9 with plasmid pBR322 DNA studied by gel electrophoresis experiments reveals that all complexes cleave DNA efficiently at micromolar concentrations under dark and anaerobic conditions probably by a hydrolytic mechanism. Complexes 3, 4, 7, 8, and [Ru(bpy)(2)(dppz)](2+) show extensive DNA cleavage in the presence of light with a shift in mobility of form I of DNA probably due to the high molecular weight of DNA-complex conjugates. However, the extent of the cleavage is augmented on irradiation in the case of complexes 3, 4, 7, and 8, which include the planar dpq and dppz ligands, suggesting a combination of hydrolytic and oxidative mechanism for the DNA scission. Molecular mechanics calculations of these systems corroborate the DNA binding and cleavage mechanisms.

Fluorescence emission and polarization for analyzing binding of ruthenium metalloglycocluster to lectin and tetanus toxin C-fragment.

We have designed and synthesized ruthenium complexes bearing clustered galactose Ru(bpy-2Gal)(3) and glucose Ru(bpy-2Glc)(3). Changes in fluorescence emission (FE) and fluorescence polarization (FP) of the metalloglycoclusters were measured by adding each lectin (peanut agglutinin (PNA), Ricinus communis agglutinin 120 (RCA), concanavalin A (ConA), or wheat germ agglutinin (WGA)) or tetanus toxin c-fragment (TCF). Following the addition of PNA and ConA, the FE spectra of Ru(bpy-2Gal)(3) and Ru(bpy-2Glc)(3) showed new emission peaks, respectively. In addition, Ru(bpy-2Gal)(3) and Ru(bpy-2Glc)(3) exclusively increased the FP values by addition of PNA and ConA. Since other combinations of the metalloglycoclusters and lectin caused little change, specific bindings of galactose to PNA and glucose to ConA were confirmed by the FE and FP measurement. From the FP analyses, the dissociation constants (K(d)) of Ru(bpy-2Gal)(3) to PNA and Ru(bpy-2Glc)(3) to ConA were calculated to be ca. 6.1 x 10(-6) M and 1.8 x 10(-5) M. Furthermore, the FP analyses proved specific binding of Ru(bpy-2Gal)(3) to TCF.

Recording of Neural Activity With Modulation of Photolysis of Caged Compounds Using Microelectrode Arrays in Rats With Seizures.

OBJECTIVE: In this paper, a new method was established to monitor multichannel neural activity with microelectrode arrays (MEAs) under modulation of caged compounds in a rat model of seizures. METHODS: The 16-channel MEAs were fabricated and implanted into the hippocampus of normal rats and epileptic rats for neural spike and local field potential (LFP) recording. Using optical fibers with drug delivery tubing, two different caged compounds [ruthenium-bipyridine-trimethylphosphine glutamate (RuBi-Glu) and ruthenium-bipyridine-trimethylphosphine gamma aminobutyric acid (RuBi-GABA)] were applied, and blue light (465 nm) was used to modulate neural activity. RESULTS: In normal rats, RuBi-Glu excited neural activity, and RuBi-GABA inhibited neural activity. The amplitude of spikes increased 26% from 154 to 194 µV with RuBi-Glu modulation. During RuBi-GABA modulation, spikes recovered to 142 µV. The firing rate increased from 1.4 to 4.5 Hz with RuBi-Glu modulation and decreased to 0.8 Hz after RuBi-GABA modulation. The power of LFPs increased from 566 to 1128 µW with RuBi-Glu modulation and recovered to 710 µW with RuBi-GABA modulation. In epileptic rats, the neural activity during seizures was significantly inhibited by RuBi-GABA modulation. The amplitude of spikes was 242 µV during seizures and decreased to 112 µV with RuBi-GABA modulation. The firing rate decreased from 20.29 to 1.33 Hz with RuBi-GABA modulation. CONCLUSION: Using MEAs, the modulation of neural activity with caged compound photolysis was observed with high temporal-spatial resolution in normal and epileptic rats. SIGNIFICANCE: This new method is important for monitoring neural activity with photo-switchable modulation.

Binding to DNA purine base and structure-activity relationship of a series of structurally related Ru(II) antitumor complexes: a theoretical study.

The thermodynamics of the binding of a series of structurally related Ru(II) antitumor complexes, that is, alpha-[Ru(azpy)2Cl2] 1, beta-[Ru(azpy)2Cl2] 2, alpha-[Ru(azpy)(bpy)Cl2] 3, and cis-[Ru(bpy)2Cl2] 4 to DNA purine bases (gunine, adenine at N7 site) has been studied by using the DFT method. The binding of imine form of 9-methyladenine (9-MeAde) to the Ru(II) moiety in a didentate fashion via its N6 and N7 atoms was also considered. The geometrical structures of the DNA model base adducts were obtained at the B3LYP/(LanL2DZ + 6-31G(d)) level in vacuo. The following exact single-point energy calculations were performed at the B3LYP/(LanL2DZ(f)+6-311+G(2d, 2p)) level both in vacuo and in aqueous solution using the COSMO model. The bond dissociation enthalpies and free energies, reaction enthalpies and free energies both in the gas phase and in aqueous solution for all considered Ru(II)-DNA model base adducts were obtained from the computations. The calculated bond dissociation enthalpies and free energies allow us to build a binding affinity order for the considered Ru(II)-DNA model base adducts. The theoretical results show that the guanine N7 is a preferred site for this series of complexes and support such an experimental fact that alpha-[Ru(azpy)(bpy)(9-EtGua)H2O](2+) (3-(9-EtGua)) is isomerized to alpha'-[Ru(azpy)(bpy)(9-EtGua)H2O](2+) (3'-(9-EtGua)). On the basis of structural and thermodynamical characteristics, the possible structure-activity relationship was obtained, and the distinct difference in cytotoxicities of this series of structurally related antitumor complexes was explained theoretically.

Engineered fungus derived FAD-dependent glucose dehydrogenase with acquired ability to utilize hexaammineruthenium(III) as an electron acceptor.

Fungal FAD-dependent glucose dehydrogenases (FADGDHs) are considered to be superior enzymes for glucose sensor strips because of their insensitivity to oxygen and maltose. One highly desirable mediator for enzyme sensor strips is hexaammineruthenium(III) chloride because of its low redox potential and high storage stability. However, in contrast to glucose oxidase (GOx), fungal FADGDH cannot utilize hexaammineruthenium(III) as electron acceptor. Based on strategic structure comparison between FADGDH and GOx, we constructed a mutant of Aspergillus flavus-derived FADGDH, capable of utilizing hexaammineruthenium(III) as electron acceptor: AfGDH-H403D. In AfGDH-H403D, a negative charge introduced at the pathway-entrance leading to the FAD attracts the positively charged hexaammineruthenium(III) and guides it into the pathway. The corresponding amino acid in wild-type GOx is negatively charged, which explains the ability of GOx to utilize hexaammineruthenium(III) as electron acceptor. Electrochemical measurements showed a response current of 46.0 µA for 10 mM glucose with AfGDH-H403D and hexaammineruthenium(III), similar to that with wild-type AfGDH and ferricyanide (47.8 µA). Therefore, AfGDH-H403D is suitable for constructing enzyme electrode strips with hexaammineruthenium(III) chloride as sole mediator. Utilization of this new, improved fungal FADGDH should lead to the development of sensor strips for blood glucose monitoring with increased accuracy and less stringent packing requirements.

Multinuclear Organometallic Ruthenium-Arene Complexes for Cancer Therapy.

There has been much recent interest in the development of therapeutic transition metal-based complexes in part fueled by the clinical success of the platinum(II) anticancer drug, cisplatin. Yet known platinum drugs are limited by their high toxicity, severe side-effects, and incidences of drug resistance. Organometallic ruthenium-arene complexes have risen to prominence as a pharmacophore due to the success of other ruthenium drug candidates in clinical trials. In this chapter, we highlight higher order multinuclear ruthenium-arene complexes and their respective investigations as chemotherapeutic agents. We discuss their unique structural properties and the associated biochemical evaluation in the context of anticancer drug design. We also review the structural considerations for the design of these scaffolds and new therapeutic applications that are uncovered for this class of complexes.

Comparative studies on DNA-binding and in vitro antitumor activity of enantiomeric ruthenium(II) complexes.

A pair of ruthenium(II) complex enantiomers, Δ- and Λ-[Ru(bpy)2PBIP]2+ {bpy=2,2'-bipyridine, PBIP=2-(4-bromophenyl)imidazo[4,5-f]1,10-phenanthroline} have been synthesized and characterized. The systematic comparative studies between two enantiomers on their DNA binding-behaviors with calf thymus DNA (CT DNA) were carried out by viscosity measurements, spectrophotometric methods and molecular simulation technology. Additional assays were performed to explore the cytotoxicity of the ruthenium(II) enantiomers against tumor cell lines. DNA-binding studies show that both the enantiomers can bind to CT DNA via intercalative mode, and the Δ form binds to CT DNA more strongly than the Λ form does. Molecular simulation further shows that both the two enantiomers intercalate between base pairs of DNA in minor groove, and that the Δ form intercalates into DNA more deeply than the Λ form does. In addition, the cell proliferation assays show that the Δ form induces a greater cytotoxicity than the Λ form on human cervical cancer HeLa cells, which is positive correlated with the results in DNA binding studies and molecular docking, and implies that the DNA binding affinities of ruthenium(II) polypyridyl complexes might be constitute to the part of their anticancer mechanisms.

In vitro and in vivo antiproliferative and trypanocidal activities of ruthenium NO donors.

BACKGROUND AND PURPOSE: Many compounds liberating NO (NO donors) have been used as therapeutic agents. Here we test two ruthenium nitrosyls, which release NO when activated by biological reducing agents, for their effects in vitro and in vivo against Trypanosoma cruzi, the agent responsible for the American trypanosomiasis (Chagas' disease). EXPERIMENTAL APPROACH: Ruthenium NO donors were incubated with a partially drug-resistant strain of T. cruzi and the anti-proliferative and trypanocidal activities evaluated. In a mouse model of acute Chagas' disease, trypanocidal activity was evaluated by measuring parasitemia, survival rate of infected mice and elimination of amastigotes in myocardial tissue. KEY RESULTS: In vitro, the observed anti-proliferative and trypanocidal activities of trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) and trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) were due to NO liberated upon reduction of these nitrosyls. Ru(NO)isn had a lower IC(50 epi) (67 microM) than the NO donor, sodium nitroprusside (IC(50 epi)=244 microM) and Ru(NO)imN (IC(50 try)=52 microM) was more potent than gentian violet (IC(50 try)=536 microM), currently used in the treatment of blood. Both ruthenium nitrosyls eliminated, in vivo, extracellular as well as intracellular forms of T. cruzi in the bloodstream and myocardial tissue and allowed survival of up to 80% of infected mice at a dose (100 nmol kg(-1) day(-1)) much lower than the optimal dose for benznidazole (385 micromol kg(-1) day(-1)). CONCLUSIONS AND IMPLICATIONS: Our data strongly suggest that NO liberated is responsible for the anti-proliferative and trypanocidal activities of the ruthenium NO donors and that these compounds are promising leads for novel and effective anti-parasitic drugs.