DNA-Bound peptide radicals generated through DNA-mediated electron transport

Flash-quench experiments were carried out to explore peptide/DNA electron-transfer reactions. DNA-bound [Ru(phen)(2)(dppz)](3+) (phen = 1,10-phenanthroline; dppz = dipyridophenazine) and [Ru(phen)(bpy')(dppz)](3+) [bpy' = 4-(4'-methyl-2, 2'-bipyridyl)valerate], generated in situ by flash-quench methodology, are powerful ground-state oxidants, capable of oxidizing guanine or tyrosine intercalated in DNA. In flash-quench experiments with mixed-sequence oligonucleotides in the presence of Lys-Tyr-Lys, transient absorption spectroscopy yielded a spectrum with a sharp maximum at 405 nm assigned to the tyrosine radical. Experiments with poly(dG.dC) suggested the intermediacy of the guanine radical, since the rise of the 405 nm signal occurred with the same kinetics as the disappearance of the guanine radical, as monitored at 510 nm. In oligonucleotide duplexes containing [Ru(phen)(bpy')(dppz)](2+) tethered at one end, damage to distant guanines was observed by gel electrophoresis, consistent with the mobility of the electron hole through the DNA duplex; the presence of the peptide did not inhibit but instead altered the distribution of guanine damage. Covalent adducts of the DNA and Lys-Tyr-Lys were detected as final irreversible products of this peptide-to-DNA electron-transfer chemistry by mass spectrometric and enzymatic digestive analysis. From these different assays and comparison of reactions of Lys-Trp-Lys and Lys-Tyr-Lys, the reactivity of the DNA-bound tyrosine radical was found to differ considerably from that of the tryptophan radical. These results establish that Lys-Tyr-Lys and Lys-Trp-Lys can participate in long-range electron-transfer reactions through the DNA from a distinct binding site. On that basis, proposals for functional roles for these peptide radicals may be considered.     

Dependence of DNA-protein cross-linking via guanine oxidation upon local DNA sequence as studied by restriction endonuclease inhibition

Oxidative damage plays a causative role in many diseases, and DNA-protein cross-linking is one important consequence of such damage. It is known that GG and GGG sites are particularly prone to one-electron oxidation, and here we examined how the local DNA sequence influences the formation of DNA-protein cross-links induced by guanine oxidation. Oxidative DNA-protein cross-linking was induced between DNA and histone protein via the flash quench technique, a photochemical method that selectively oxidizes the guanine base in double-stranded DNA. An assay based on restriction enzyme cleavage was developed to detect the cross-linking in plasmid DNA. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were then used to probe the plasmid surface for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA-protein cross-linking, whereas the AT-recognizing enzyme AseI was unaffected in its cleavage. These experiments also reveal the susceptibility of different guanine sites toward oxidative cross-linking. The percent inhibition observed for the endonucleases, and their pBR322 cleavage sites, decreased in the order: PpuMI (5'-GGGTCCT-3' and 5'-AGGACCC-3') > BamHI (5'-GGATCC-3') > EcoRI (5'-GAATTC-3'), a trend consistent with the observed and predicted tendencies for guanine to undergo one-electron oxidation: 5'-GGG-3' > 5'-GG-3' > 5'-GA-3'. Thus, it appears that in mixed DNA sequences the guanine sites most vulnerable to oxidative cross-linking are those that are easiest to oxidize. These results further indicate that equilibration of the electron hole in the plasmid DNA occurs on a time scale faster than that of cross-linking.     

The relationship between the structures of periphery ligands and the DNA binding mode of [Ru(II)(1,10-phenanthroline)(L1L2)dipyrido[3,2-a:2',3'-c]phenazine](n+) (L1=Cl or pyridine and L2=pyridine, n=1,2)

The binding modes of the [Ru(II)(1,10-phenanthroline)(L₁L₂) dipyrido[3,2-a:2′,3′-c]phenazine]²⁺ {[Ru(phen)(py) Cl dppz]⁺ (L₁ = Cl, L₂ = pyridine) and ([Ru(phen)(py)₂dppz]²⁺ (L₁ = L₂ = pyridine)} to native DNA is compared to that of the [Ru(II)(1,10-phenanthroline)₂dipyrido[3,2-a:2′,3′-c]phenazine]²⁺ complex ([Ru(phen)₂dppz]²⁺) by various spectroscopic and hydrodynamic methods including electric absorption, linear dichroism (LD), fluorescence spectroscopy, and viscometric titration. All measured properties, including red-shift and hypochromism in the dppz absorption band, nearly perpendicular molecular plane of the dppz ligand with respect to the local DNA helix axis, prohibition of the ethidium binding, the light switch effect and binding stoichiometry, increase in the viscosity upon binding to DNA, increase in the melting temperature are in agreement with classical intercalation of dppz ligand of the [Ru(phen)₂dppz]²⁺ complex, in which both phenanthroline ligand anchored to the DNA phosphate groups by electrostatic interaction. [Ru(phen)(py)₂ dppz]²⁺ and [Ru(phen)(py) Cl dppz]⁺ complexes had one of the phenanthroline ligand replaced by either two pyridine ligands or one pyridine plus a chlorine ion. They exhibited similar protection from water molecules, interaction with DNA bases, and occupying site that is common with ethidium. The dppz ligand of these two Ru(II) complex were greatly tilted relative to the DNA helix axis, suggesting that the dppz ligand resides inside the DNA and is not perpendicular relative to the DNA helix axis. These observation suggest that anchoring the [Ru(phen)₂dppz]²⁺complex by both phenanthroline is essential for the dppz ligand to be classically intercalated between DNA base-pairs.     

Picosecond Kerr-gated time-resolved resonance Raman spectroscopy of the [Ru(phen)(2)dppz](2+) interaction with DNA

To investigate the basis of the 'light-switch' effect, the solvent dependence of the Kerr-gated picosecond-time resolved resonance Raman (TR(3)) spectra of [Ru(bpy)(2)dppz](2+), [Ru(phen)(2)dppz](2+), and the modified complex [Ru(phen)(2)cpdppzOMe](2+) and a dimer [mu-C4(cpdppz)(2)-(phen)(4)Ru(2)](4+) were studied. The investigation focussed on comparing the behaviour of [Ru(phen)(2)dppz](2+) in acetonitrile, ethanol, H(2)O, D(2)O, and DNA. The data are consistent with a model wherein excitation induces metal-to-ligand charge transfer (MLCT) to any of the ligands (termed the 'precursor' state) which, by interligand electron transfer (ILET), produces an excited state localised on the dppz ligand, MLCT(1). In water this state relaxes with a characteristic time of approximately 6 ps to a non-emissive state (MLCT(2)). The TR(3) spectra in water, acetonitrile and DNA are all distinctly different. However, the early (4 ps) water spectrum resembles the spectrum in DNA. This interesting observation suggests that the DNA-bound excited state of the complex can be thought of as a model for the initial, poorly solvated state in water.     

Synthesis, crystal structure, DNA binding and photo-induced DNA cleavage activity of (S-methyl-L-cysteine)copper(II) complexes of heterocyclic bases

Ternary S-methyl-L-cysteine (SMe-l-cys) copper(II) complexes [Cu(SMe-L-cys)(B)(H(2)O)](X) (1-4), where the heterocyclic base B is 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), dipyridoquinoxaline (dpq, 3) and dipyridophenazine (dppz, 4), and X is ClO(4)(-) (1-3) or NO(3)(-) (4), are prepared and their DNA binding and cleavage properties studied. Complexes 2 and 4 are structurally characterized by X-ray crystallography. Both the crystal structures show distorted square-pyramidal (4+1) CuN(3)O(2) coordination geometry of the complexes in which the N,O-donor S-methyl-L-cysteine and N,N-donor heterocyclic base bind at the basal plane with a water molecule as the axial ligand. In addition, the dppz structure shows the presence of a 1D-chain formed due to covalent linkage of the carboxylate oxygen atom belonging to another molecule at the elongated axial site. The crystal structures show chemically significant non-covalent interactions like hydrogen bonding involving the axial aqua ligand and pi-pi interactions between dppz ligands. The complexes display a d-d band in the range of 605-654 nm in aqueous dimethylformamide (DMF) solution (9:1 v/v). The redox active complexes show quasireversible cyclic voltammetric response near 0.1 V in DMF assignable to the Cu(II)/Cu(I) couple. The complexes show good binding affinity to calf thymus (CT) DNA giving the order: 4 (dppz)>3 (dpq)>2 (phen)>1 (bpy). The intrinsic binding constants, obtained from UV-visible spectroscopic studies, are 1.3x10(4) and 2.15 x 10(4) M(-1) for 3 and 4, respectively. Control DNA cleavage experiments using pUC19 supercoiled (SC) DNA and minor groove binder distamycin suggest major groove binding propensity for the dppz complex, while the phen and dpq complexes bind at the minor groove of DNA. Complexes 2-4 show DNA cleavage activity in dark in the presence of a reducing agent 3-mercaptopropionic acid (MPA) via a mechanistic pathway involving formation of hydroxyl radical as the reactive species. The complexes also show efficient photo-induced DNA cleavage activity on irradiation with a monochromatic UV light of 365 nm in absence of any external reagent. The cleavage efficiency follows the order: 3>4>2. The complexes exhibit significant DNA cleavage activity on irradiation with visible light of 633 nm. Control experiments show inhibition of cleavage in presence of singlet oxygen quenchers like sodium azide, histidine and enhancement of cleavage in D(2)O, suggesting formation of singlet oxygen as a reactive species in a type-II process. The photosensitizing effect of the thiomethyl group of the amino acid is evidenced from the observation of significant DNA photocleavage activity of the phen complex 2 as the phen ligand itself is not a photosensitizer.     

DNA cross-linking with metallointercalator-peptide conjugates

Short peptides have been tethered to a DNA-intercalating ruthenium complex to create a photoactivated cross-linking reagent. The ruthenium complex, [Ru(phen)(bpy')(dppz)]2+ (phen = 1,10-phenanthroline, bpy' = 4-(butyric acid)-4'-methyl-2,2'-bipyridine, and dppz = dipyridophenazine), delivers the peptide to DNA and initiates the cross-linking reaction by oxidizing DNA upon irradiation in the presence of an oxidative quencher. The tethered peptide, only five to six residues in length, forms cross-links with the oxidized site in DNA. Cross-linking was detected and studied by gel electrophoresis and through spectroscopic measurements. The ruthenium-peptide complex is luminescent when bound to DNA, and the binding constants for several intercalator-peptide conjugates were determined by luminescence titration. The composition of the peptide affects both binding affinity and the extent of cross-linking. The greatest amounts of cross-linking were observed with tethered peptides that contained positively charged residues, either lysine or arginine. To test the impact of individual residues on cross-linking, the central residue in a 5-mer peptide was substituted with seven different amino acids. Though mutation of this position had only a small effect on the extent of cross-linking, it was discovered that peptides containing Trp or Tyr gave a distinctive pattern of products in gels. In experiments using the untethered peptide and ruthenium complex, it was determined that delivery of the peptide by the ruthenium intercalator is not essential for cross-linking; peptide attachment to the metal complex can constrain cross-linking. Importantly, the cross-linking adducts produced with ruthenium-peptide conjugates are luminescent and thus provide a luminescent cross-linking probe for DNA.     

Characterization of dipyridophenazine complexes of ruthenium(II): the light switch effect as a function of nucleic acid sequence and conformation.

Spectroscopic parameters for two novel ruthenium complexes on binding to nucleic acids of varying sequences and conformations have been determined. These complexes, Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline; dppz = dipyrido[3,2:a-2',3':c]-phenazine) serve as "molecular light switches" for DNA, displaying no photoluminescence in aqueous solution but luminescing intensely in the presence of DNA. The luminescent enhancement observed upon binding is attributed to the sensitivity of the excited state to quenching by water; in DNA, the metal complex, upon intercalation into the helix, is protected from the aqueous solvent, thereby preserving the luminescence. Correlations between the extent of protection (depending upon the DNA conformation) and the luminescence parameters are observed. Indeed, the strongest luminescent enhancement is observed for intercalation into DNA conformations which afford the greatest amount of overlap with access from the major groove, such as in triple helices. Differences are observed in the luminescent parameters between the two complexes which also correlate with the level of water protection. In the presence of nucleic acids, both complexes exhibit biexponential decays in emission. Quenching studies are consistent with two intercalative binding modes for the dppz ligand from the major groove: one in which the metal-phenazine axis lies along the DNA dyad axis and another where the metal-phenazine axis lies almost perpendicular to the DNA dyad axis. Ru(bpy)2dppz2+ and Ru(phen)2dppz2+ are shown here to be unique reporters of nucleic acid structures and may become valuable in the design of new diagnostics for DNA.     

DNA binding by Ru(II)–bis(bipyridine)–pteridinyl complexes

The interactions of five bis(bipyridyl) Ru(II) complexes of pteridinyl-phenanthroline ligands with calf thymus DNA have been studied. The pteridinyl extensions were selected to provide hydrogen-bonding patterns complementary to the purine and pyrimidine bases of DNA and RNA. The study includes three new complexes [Ru(bpy)(2)(L-pterin)](2+), [Ru(bpy)(2)(L-amino)](2+), and [Ru(bpy)(2)(L-diamino)](2+) (bpy is 2,2'-bipyridine and L-pterin, L-amino, and L-diamino are phenanthroline fused to pterin, 4-aminopteridine, and 2,4-diaminopteridine), two previously reported complexes [Ru(bpy)(2)(L-allox)](2+) and [Ru(bpy)(2)(L-Me(2)allox)](2+) (L-allox and L-Me(2)allox are phenanthroline fused to alloxazine and 1,3-dimethyalloxazine), the well-known DNA intercalator [Ru(bpy)(2)(dppz)](2+) (dppz is dipyridophenazine), and the negative control [Ru(bpy)(3)](2+). Reported are the syntheses of the three new Ru-pteridinyl complexes and the results of calf thymus DNA binding experiments as probed by absorption and fluorescence spectroscopy, viscometry, and thermal denaturation titrations. All Ru-pteridine complexes bind to DNA via an intercalative mode of comparable strength. Two of these four complexes-[Ru(bpy)(2)(L-pterin)](2+) and [Ru(bpy)(2)(L-allox)](2+)-exhibit biphasic DNA melting curves interpreted as reflecting exceptionally stable surface binding. Three new complexes-[Ru(bpy)(2)(L-diamino)](2+), [Ru(bpy)(2)(L-amino)](2) and [Ru(bpy)(2)(L-pterin)](2+)-behave as DNA molecular "light switches."     

Syntheses and DNA-binding studies of two ruthenium(II) complexes containing one ancillary ligand of bpy or phen: [Ru(bpy)(pp[2,3]p)2](ClO4)2 and [Ru(phen)(pp[2,3]p)2](ClO4)2

Two new ruthenium(II) complexes of [Ru(bpy)(pp[2,3]p)2](ClO4)2 and [Ru(phen)(pp[2,3]p)2](ClO4)(2) (bpy=2,2'-bipyridine, phen=1,10-phenanthroline, pp[2,3]p=pyrido[2',3':5,6]pyrazino[2,3-f][1,10]phenanthroline) have been synthesized and characterized by elemental analysis and 1H NMR spectra. The calf thymus DNA-binding properties of the two complexes were investigated by UV-visible and emission spectroscopy, competitive binding experiments with ethidium bromide and viscosity measurements. The results indicate that the two complexes intercalate between the base pairs of the DNA tightly with intrinsic DNA-binding constants of 3.08 x 10(6) and 6.53 x 10(6) M(-1) in buffered 50 mM NaCl, respectively, which are much larger than 6.9 x 10(5) M(-1) for [Ru(bpy)2(pp[2,3]p)](ClO4)2 containing two ancillary ligands of bpy.     

Ruthenium(II)-phenanthroline-biotin complexes: synthesis and luminescence enhancement upon binding to avidin

We report the synthesis, characterization, and avidin-binding properties of two novel ruthenium complexes, [Ru(bpy)(2)(phen-biotin)][PF(6)](2) 1 and [Ru(phen)(2)(phen-biotin)][PF(6)](2) 2 (bpy = 2,2'-bipyridine; phen = 1,10-phenanthroline, phen-biotin = 5-(10-amidobiotinyl)-1,10-phenanthroline)). We demonstrate that both biotinylated compounds bind to avidin through their biotin moieties with high affinity and in a 4:1 ratio. The binding of compounds 1 and 2 to avidin results in an enhancement in luminescence intensity ( approximately 1.4x, approximately 1.6x, respectively), relative to the unbound biotinylated ruthenium complexes. This behavior is markedly different from biotinylated organic dyes, whose fluorescence is quenched upon binding to avidin. Thus, ruthenium-biotin complexes 1 and 2 can form the basis of new, simplified biotin-avidin assays, which involve luminescence detection of the relevant biotinylated molecule through cross-linking with avidin.     

Development of a novel enzyme-redox-mediator system based on a fungal laccase and ruthenium complexes

The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)2]PF6 and Ru(bpy)2CO3 under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)2]PF6 and 3 for Ru(bpy)2CO3. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.     

[Ru(phen)2(dppz)] as an efficient optical probe for staining nuclear components

The ‘molecular light switch’ complexes [Ru(bpy)₂(dppz)]²⁺ (1) and [Ru(phen)₂(dppz)]²⁺ (2), where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline and dppz = dipyrido[3,2-a:2′,3′-c]phenazine, have been explored as probes for diagnosing and staining nuclear components. The phen complex acts as a better staining agent for nonviable cells than for viable cells and exhibits a staining efficiency in tail region of comet more specific and stronger than the already known dye Hoechst 33258.     

Photo-induced oxidation of a dinuclear Mn(2)(II,II) complex to the Mn(2)(III,IV) state by inter- and intramolecular electron transfer to Ru(III)tris-bipyridine

To model the structural and functional parts of the water oxidizing complex in Photosystem II, a dimeric manganese(II,II) complex (1) was linked to a ruthenium(II)tris-bipyridine (Ru(II)(bpy)(3)) complex via a substituted L-tyrosine, to form the trinuclear complex 2 [J. Inorg. Biochem. 78 (2000) 15]. Flash photolysis of 1 and Ru(II)(bpy)(3) in aqueous solution, in the presence of an electron acceptor, resulted in the stepwise extraction of three electrons by Ru(III)(bpy)(3) from the Mn(2)(II,II) dimer, which then attained the Mn(2)(III,IV) oxidation state. In a similar experiment with compound 2, the dinuclear Mn complex reduced the photo-oxidized Ru moiety via intramolecular electron transfer on each photochemical event. From EPR it was seen that 2 also reached the Mn(2)(III,IV) state. Our data indicate that oxidation from the Mn(2)(II,II) state proceeds stepwise via intermediate formation of Mn(2)(II,III) and Mn(2)(III,III). In the presence of water, cyclic voltammetry showed an additional anodic peak beyond Mn(2)(II,III/III,III) oxidation which was significantly lower than in neat acetonitrile. Assuming that this peak is due to oxidation to Mn(2)(III,IV), this suggests that water is essential for the formation of the Mn(2)(III,IV) oxidation state. Compound 2 is a structural mimic of the water oxidizing complex, in that it links a Mn complex via a tyrosine to a highly oxidizing photosensitizer. Complex 2 also mimics mechanistic aspects of Photosystem II, in that the electron transfer to the photosensitizer is fast and results in several electron extractions from the Mn moiety.     

Highly sensitive and selective photoelectrochemical DNA sensor for the detection of Hg²⁺ in aqueous solutions

A "turn-on" photoelectrochemical sensor for Hg(2+) detection based on thymine-Hg(2+)-thymine interaction is presented by using a thymine-rich oligonucleotide film and a double-strand DNA intercalator, Ru(bpy)(2)(dppz)(2+) (bpy=2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c]phenazine) as the photocurrent signal reporter. The presence of Hg(2+) induces the formation of a double helical DNA structure which provides binding sites for Ru(bpy)(2)(dppz)(2+). The double helical structure was confirmed by circular dichroism and fluorescence measurements. Under the optimized conditions, a linear relationship between photocurrent and Hg(2+) concentration was obtained over the range of 0.1 nM to 10 nM Hg(2+), with a detection limit of 20 pM. Interference by 10 other metal ions was negligible. Analytical results of Hg(2+) spiked into tap water and lake water by the sensor were in good agreement with mass spectrometry data. With the advantages of high sensitivity and selectivity, simple sensor construction, low instrument cost and low sample volume, this method is potentially suitable for the on-site monitoring of Hg(2+) contamination.     

Development of a novel enzyme-redox-mediator system based on a fungal laccase and ruthenium complexes

The laccase produced by the fungus Coriolus hirsutus has been coordinatively modified with ruthenium complexes [Ru(phpy)(phen)(MeCN)₂]PF₆ and Ru(bpy)₂CO₃ under aerobic and anaerobic conditions. The amount of the complexes per enzyme molecule does not depend on the oxygen concentration, equaling 5 for [Ru(phpy)(phen)(MeCN)₂]PF₆ and 3 for Ru(bpy)₂CO₃. The pH dependence of the enzymatic activity, thermostability, and catalytical and electrocatalytical properties of the modified laccase are reported. It has been shown that, during the modification, at least one molecule of the ruthenium compound was coordinated near the T1 active center of the laccase, being directly involved in the catalysis and enhancing its efficiency.     

Luminescence of ruthenium(II) polypyridyls: evidence for intercalative binding to Z-DNA.

Photophysical studies have been undertaken to characterize the binding interactions of enantiomers of Ru(phen)3(2+), Ru(DIP)3(2+), and racemic Ru(bpy)2dppz2+ (where phen = 1,10-phenanthroline, DIP = 4,7-diphenylphenanthroline, and dppz = dipyridophenazine) with Z-form poly d(GC). Parallel enhancements in steady state luminescent intensity and a lengthening of luminescent lifetimes are seen for ruthenium enantiomers with Z-DNA as for B-DNA but with enantioselectivities reversed. Greater enhancements are seen for delta-isomers with the right-handed helix but for lambda-isomers with the left-handed helix. Ru(bpy)2dppz2+, an avid intercalator in B-DNA, displays no luminescence free in aqueous solution, but luminesces brightly bound to either B- or Z-poly d(GC). Stern-Volmer quenching studies also support the enantioselective preference in binding to B-DNA by delta-isomers and a reversal with binding to Z-DNA preferentially by the lambda-isomers. Steady state polarization studies indicate a rigid association of the complexes with both B- and Z-DNA on the time-scale of their emission and again with symmetrical enantioselectivities for the left and right-handed helices. Given the well characterized intercalative association of the complexes with B-DNA, the parallel results seen here with Z-DNA point strongly to a comparable intercalative association with the Z-form helix. That molecules may interact with Z-DNA through intercalation has not been demonstrated previously and now requires consideration in describing the range of interactions of small molecules and proteins with Z-DNA.     

Cyclometalated ruthenium(II) complexes of benzo[h]quinoline (bzqH)[Ru(bzq)(NCMe)4], [Ru(bzq)(LL)(NCMe)2], and [Ru(bzq)(LL)2] (LL = bpy, phen)

Cyclometalation of benzo[h]quinoline (bzqH) by [RuCl(μ-Cl)(η⁶-C₆H₆)]₂ in acetonitrile occurs in a similar way to that of 2-phenylpyridine (phpyH) to afford [Ru(bzq)(MeCN)₄]PF₆ (3) in 52% yield. The properties of 3 containing ‘non-flexible’ benzo[h]quinoline were compared with the corresponding [Ru(phpy)(MeCN)₄]PF₆ (1) complex with ‘flexible’ 2-phenylpyridine. The [Ru(phpy)(MeCN)₄]PF₆ complex is known to react in MeCN solvent with ‘non-flexible’ diimine 1,10-phenanthroline to form [Ru(phpy)(phen)(MeCN)₂]PF₆, being unreactive toward ‘flexible’ 2,2′-bipyridine under the same conditions. In contrast, complex 3 reacts both with phen and bpy in MeCN to form [Ru(bzq)(LL)(MeCN)₂]PF₆ {LL = bpy (4) and phen (5)}. Similar reaction of 3 in methanol results in the substitution of all four MeCN ligands to form [Ru(bzq)(LL)₂]PF₆ {LL = bpy (6) and phen (7)}. Photosolvolysis of 4 and 5 in MeOH occurs similarly to afford [Ru(bzq)(LL)(MeCN)(MeOH)]PF₆ as a major product. This contrasts with the behavior of [Ru(phpy)(LL)(MeCN)₂]PF₆, which lose one and two MeCN ligands for LL = bpy and phen, respectively. The results reported demonstrate a profound sensitivity of properties of octahedral compounds to the flexibility of cyclometalated ligand. Analogous to the 2-phenylpyridine counterparts, compounds 4-7 are involved in the electron exchange with reduced active site of glucose oxidase from Aspergillus niger. Structure of complexes 4 and 6 was confirmed by X-ray crystallography.     

Different modes of DNA cleavage activity of dihydroxo-bridged dicopper(II) complexes having phenanthroline bases

Dihydroxo-bridged dicopper(II) complexes [(Cu(phen))(2)(mu-OH)(2)](ClO(4))(2) (1), [(Cu(dpq))(2)(mu-OH)(2)](ClO(4))(2) (2) and [(Cu(dppz)(DMF))(2)(mu-OH)(2)](PF(6))(2) (3), where phen, dpq and dppz are 1,10-phenanthroline, dipyridoquinoxaline and dipyridophenazine, respectively, are prepared and their DNA binding and cleavage properties studied. Complex 3 has been structurally characterized by X-ray crystallography. The complexes have a (Cu(2)(mu-OH)(2))(2+) core with an essentially planar arrangement of two CuN(2)O(2) basal planes. The complexes are avid binder to calf thymus DNA (K(app) value of 4.8 x 10(6) and 5.9 x 10(6) M(-1) for 2 and 3, respectively, from ethidium displacement assay) and exhibits significant cleavage of supercoiled (SC) pUC19 DNA in dark in presence of mercaptopropionic acid. Besides, the dpq and dppz complexes display photo-induced DNA cleavage on UV (312 nm) and red light (632.8 nm) irradiations in absence of any additives. Mechanistic investigations reveal minor groove binding for the phen and dpq complexes, and major groove preference for the dppz species. The oxidative DNA cleavage reactions in presence of mercaptopropionic acid as a reducing agent involve hydroxyl radicals. The photo-cleavage reactions at UV light involve singlet oxygen as the reactive species, while similar reactions on red light irradiation (632.8 nm) proceed through the formation of hydroxyl radical. The complexes show significant DNA hydrolase activity in absence of any additives under dark reaction conditions.     

A novel phenotype-based approach for systematically screening antiproliferation metallodrugs

Ruthenium (Ru) derivatives have less toxicity and higher water-solubility than cisplatin, giving them great potential as antitumor metallodrugs. In this study, zebrafish were employed as a whole-organism model to screen new Ru compounds for anti-cell proliferation activity. After soaking fish embryos in cisplatin and five Ru derivatives, [Ru(terpy)(bpy)Cl]Cl, [Ru(terpy)(dppz)OH₂](ClO₄)₂, [Ru(terpy)(tMen)OH₂](ClO₄)₂, [Ru(terpy)(Me₄Phen)OH₂](ClO₄)₂, and Ru(bpy)₂Cl₂, only cisplatin and [Ru(terpy)(bpy)Cl]Cl-treated embryos displayed obvious phenotypic effects, such as fin-reduction. After further modification of [Ru(terpy)(bpy)Cl]Cl's main structure and the synthesis of two structurally related compounds, [Ru(terpy)(dcbpyH₂)Cl]Cl and [Ru(terpy)(dmbpy)Cl]Cl, only [Ru(terpy)(dmbpy)Cl]Cl exhibited fin-reduction phenotypes. TUNEL assays combined with immunostaining techniques revealed that treatment with cisplatin, [Ru(terpy)(bpy)Cl]Cl, and [Ru(terpy)(dmbpy)Cl]Cl led proliferating fin mesenchymal cells to undergo apoptosis and consequently caused fin-reduction phenotypes. Furthermore, [Ru(terpy)(bpy)Cl]Cl was able to activate the P53-dependent and independent pathways, and induced human hepatoma cells to undergo apoptosis. In summary, it was concluded that the zebrafish model was effective for the screening of phenotype-based antiproliferation metallodrugs.     

Electrochemiluminescence of dipicolinic acid (DPA) and (bpy)(2)Ru(DPA)(+) (bpy = 2,2'-bipyridine).

The spectroscopic and electrochemiluminescence (ECL) properties of dipicolinic acid (DPA), (bpy)(2)Ru(2+) (bpy = 2,2'-bipyridine) and the species formed when DPA and (bpy)(2)Ru(2+) [abbreviated to (bpy)(2)Ru(DPA)(+)] are allowed to react are reported. The UV-Vis absorption maxima for (bpy)(2)Ru(2+) and (bpy)(2)Ru(DPA)(+) are 493 and 475 nm, respectively, indicating the in situ formation of a complex between DPA and (bpy)(2)Ru(2+). DPA, (bpy)(2)Ru(2+) and (bpy)(2)Ru(DPA)(+) display ECL upon oxidation in the presence of the oxidative-reductive co-reactant tri-n-propylamine (TPrA). The ECL of (bpy)(2)Ru(DPA)(+) is at least two-fold higher than either of the parent species. An ECL spectrum of (bpy)(2)Ru(DPA)(+) displays a peak maximum 40 nm red-shifted from the photoluminescence peak maximum, suggesting that the excited state formed electrochemically is different from that formed spectroscopically.