Ruthenium Dipyridophenazine Research Articles

Deconvoluting binding sites in amyloid nanofibrils using time-resolved spectroscopy.

Steady-state fluorescence spectroscopy has a central role not only for sensing applications, but also in biophysics and imaging. Light switching probes, such as ruthenium dipyridophenazine complexes, have been used to study complex systems such as DNA, RNA, and amyloid fibrils. Nonetheless, steady-state spectroscopy is limited in the kind of information it can provide. In this paper, we use time-resolved spectroscopy for studying binding interactions between amyloid-β fibrillar structures and photoluminescent ligands. Using time-resolved spectroscopy, we demonstrate that ruthenium complexes with a pyrazino phenanthroline derivative can bind to two distinct binding sites on the surface of fibrillar amyloid-β, in contrast with previous studies using steady-state photoluminescence spectroscopy, which only identified one binding site for similar compounds. The second elusive binding site is revealed when deconvoluting the signals from the time-resolved decay traces, allowing the determination of dissociation constants of 3 and 2.2 μM. Molecular dynamic simulations agree with two binding sites on the surface of amyloid-β fibrils. Time-resolved spectroscopy was also used to monitor the aggregation of amyloid-β in real-time. In addition, we show that common polypyridine complexes can bind to amyloid-β also at two different binding sites. Information on how molecules bind to amyloid proteins is important to understand their toxicity and to design potential drugs that bind and quench their deleterious effects. The additional information contained in time-resolved spectroscopy provides a powerful tool not only for studying excited state dynamics but also for sensing and revealing important information about the system including hidden binding sites.

Localizing the initial excitation – A case study on NiO photocathodes using Ruthenium dipyridophenazine complexes as sensitizers

We report on the localization of the initially excited electronic state within the molecular framework of a series of [Ru(bpy)2dppz]2+ derivatives (bpy:2,2′-bipyridine, dppz: dipyrido-phenazine) as sensitizers in NiO based photocathodes. The introduction of conjugated linkers with phenylene and triazole moieties in the bpy ligand sphere separates the NiO surface from the metal center and hence is considered to stabilize the charge separated state, which results from light-driven hole injection. However, introduction of the conjugated linkers also alters the localization of the excess electron density in the excited state within the ligand sphere and impacts the extent to which the charge-separated state is formed. The study emphasizes that tuning the ligand with the lowest-energy π* orbital distal or proximal to the NiO surface significantly affects the initial charge-separation and the solar cell performance. The stability of the charge-separated state correlates with the observed photocurrents in dye-sensitized solar cells. Furthermore, the study challenges the widely accepted concept that the introduction of extended anchoring groups, i.e. increasing Ru – NiO distance, stabilizes the charge-separated state and suppresses charge recombination at the metal-oxide molecule interface.

Two Emissive Long-Lived Excited States of an Imidazole-Functionalized Ruthenium Dipyridophenazine Complex

A ruthenium(II) polypyridine-type complex based on the dipyridophenazine ligand with a directly fused imidazole unit (L1, dipyrido[3,2-a:2',3'-c]phenazine-10,11-imidazole) has been synthesized, and its electrochemical and photophysical properties have been studied. The cyclic voltammogram of [Ru(tbbpy)2(L1)]2+ (C1) (tbbpy is 4,4'-tert-butyl-2,2'-bipyridine) shows a cathodic shift of the phenazine-based reduction process compared to similar molecules, while the first detected reduction wave (-1.34 V vs Fc/Fc+) is assigned to the imidazole unit within the molecule. On the basis of the TD-DFT calculations, the strong visible absorption band exhibited by C1 is assigned to a metal-to-ligand charge transfer (MLCT) transition with a concurrent ligand-centered (LC) transition. At room-temperature, C1 features emission (Φ = 0.04) from its lowest excited states with time constants of 1.2 and 18.3 μs. These lifetimes are assigned to emission processes from the 3MLCT and 3LC state, respectively. This is the first time that a long-lived dual emission has been observed for a ruthenium(II) complex bearing a directly fused extended π-system. Furthermore, the emission of C1 is quenched upon water addition. In contrast to related compounds based on a dipyridophenazine ligand, the excited state energy is not shifted, and the lifetime is drastically decreased to 169 ns.

Metal complexes and time-resolved photoluminescence spectroscopy for sensing applications

This feature article will cover our efforts to sense biologically relevant molecules using photoluminescent metal complexes. Photoluminescent metal complexes possess large Stokes shifts, long lifetimes and tunable photoluminescence maxima. We have developed probes for DNA and RNA detection containing metal complexes of ruthenium and iridium as photoluminescent reporters. Iridium complexes have also been modified to serve as probes for amino acids such as cysteine, homocysteine, and histidine. In addition, probes sensible to the aggregation state of proteins such as amyloid-β and alpha-synuclein were developed, which display changes in photoluminescence (ruthenium dipyridophenazine complexes) or birefringence (ruthenium red). Finally, we will present the detection of solvent vapors using a photoluminescent rhenium complex within a zeolite matrix. The long photoluminescence lifetime of the aforementioned complexes has been synergistically combined with advanced time-resolved methods to enhance the detection scope of these probes. For example techniques such as time-gating have been used to detect oligonucleotides, amino acids and protein aggregation even in highly autofluorescent media. Time-gating allows selecting a time-window in a time-resolved emission spectrum where the long-lived photoluminescence of the probes can be preferentially detected from the short-lived autofluorescence of the medium. In addition, we have used time-resolved photoluminescence spectroscopy to extract the photoluminescence of free histidine from the photoluminescence of histidine-containing proteins. Also, a combination of photoluminescence intensity, maximum and lifetime was used to detect solvent vapors. These examples testify on the advantages of time-resolved photoluminescence spectroscopy for enhancing the detection of analytes using probes with long-lived photoluminescence.

Structure determination of an intercalating ruthenium dipyridophenazine complex which kinks DNA by semiintercalation of a tetraazaphenanthrene ligand

We describe a crystal structure, at atomic resolution (1.1 Å, 100 K), of a ruthenium polypyridyl complex bound to duplex DNA, in which one ligand acts as a wedge in the minor groove, resulting in the 51° kinking of the double helix. The complex cation Λ-[Ru(1,4,5,8-tetraazaphenanthrene)(2)(dipyridophenazine)](2+) crystallizes in a 11 ratio with the oligonucleotide d(TCGGCGCCGA) in the presence of barium ions. Each complex binds to one duplex by intercalation of the dipyridophenazine ligand and also by semiintercalation of one of the orthogonal tetraazaphenanthrene ligands into a second symmetrically equivalent duplex. The result is noncovalent cross-linking and marked kinking of DNA.

Ruthenium(II) Complex Enantiomers as Cellular Probes for Diastereomeric Interactions in Confocal and Fluorescence Lifetime Imaging Microscopy

Ruthenium dipyridophenazine (dppz) complexes are sensitive luminescent probes for hydrophobic environments. Here, we apply multiple-frequency fluorescence lifetime imaging microscopy (FLIM) to Δ and Λ enantiomers of lipophilic ruthenium dppz complexes in live and fixed cells, and their different lifetime staining patterns are related to conventional intensity-based microscopy. Excited state lifetimes of the enantiomers determined from FLIM measurements correspond well with spectroscopically measured emission decay curves in pure microenvironments of DNA, phospholipid membrane or a model protein. We show that FLIM can be applied to monitor the long-lived excited states of ruthenium complex enantiomers and, combined with confocal microscopy, give new insight into their biomolecular binding and reveal differences in the microenvironment probed by the complexes.

Correlation Between Cellular Localization and Binding Preference to RNA, DNA, and Phospholipid Membrane for Luminescent Ruthenium(II) Complexes

Because of their unique photophysical properties, sensitively depending on environment, ruthenium dipyridophenazine (dppz) complexes are interesting as probes for cellular imaging with fluorescence microscopy. Here three complexes derivatized with alkyl ether chains of varied length, which exhibit distinctly different cellular staining patterns by confocal laser scanning microscopy, are studied regarding their binding preference for rRNA compared with calf thymus DNA (ct-DNA) and phospholipid membranes. Co-staining with commercial RNA and membrane-specific dyes shows that whereas the least lipophilic complex exclusively stains DNA inside the nucleus, the most lipophilic complex preferentially stains membrane-rich parts of the cell. Interestingly, only the intermediate lipophilic complex shows intense staining of the RNA-rich nucleoli. The intracellular localizations of the probes correlate with their binding preferences concluded from spectroscopy measurements.

Lipophilic ruthenium complexes with tuned cell membrane affinity and photoactivated uptake

Ruthenium dipyridophenazine (dppz) complexes are virtually non-emissive in aqueous solutions but show strong luminescence in hydrophobic environments, making them interesting as molecular probes in cellular imaging. We show by luminescence spectroscopy that by substituting the dppz ligand with alkyl ether chains of increasing length the complexes can be tuned from preferential intercalation into DNA to insertion in model phospholipid membranes. Confocal laser scanning microscopy (CLSM) on methanol fixed CHO-K1 cells show an analogous distribution in the cell, where the least hydrophobic complex exclusively stains the nucleus whereas the more hydrophobic ones seem to predominantly stain membrane structures in the cytoplasm. In live cells CLSM show that initially only the more hydrophobic derivatives stain the plasma membrane. However, brief further exposure to the laser light causes permeabilization of the membrane and accumulation of extracellular ruthenium complexes in internal cellular structures, similarly to the distribution found in fixed cells.

Photooxidation of Guanine by a Ruthenium Dipyridophenazine Complex Intercalated in a Double‐Stranded Polynucleotide Monitored Directly by Picosecond Visible and Infrared Transient Absorption Spectroscopy

Transient species formed by photoexcitation (400 nm) of [Ru(dppz)(tap)2]2+ (1) (dppz = dipyrido[3,2-a:2',3'-c]phenazine; tap=1,4,5,8-tetraazaphenanthrene) in aqueous solution and when intercalated into a double-stranded synthetic polynucleotide, [poly(dG-dC)]2, have been observed on a picosecond timescale by both visible transient absorption (allowing monitoring of the metal complex intermediates) and transient infrared (IR) absorption spectroscopy (allowing direct study of the DNA nucleobases). By contrast with its behavior when free in aqueous solution, excitation of 1 when bound to [poly(dG-dC)]2 causes a strong increase in absorbance at 515 nm due to formation of the reduced complex [Ru(dppz)(tap)2]+ (rate constant=(2.0+/-0.2) x 10(9) s(-1)). The subsequent reformation of 1 proceeds with a rate constant of (1.1+/-0.2) x 10(8) s(-1). When the process is carried out in D2O, the rates of formation and removal of [Ru(dppz)(tap)2]+ are reduced (rate constants (1.5+/-0.3) x 10(9) and (0.7+/-0.2) x 10(8) s(-1) respectively) consistent with proton-coupled electron transfer processes. Picosecond transient IR measurements in the 1540-1720 cm(-1) region in D2O solution confirm that the reduction of 1 intercalated into [poly(dG-dC)]2 is accompanied by bleaching of IR ground-state bands of guanine (1690 cm(-1)) and cytosine (1656 cm(-1)), each with similar rate constants.

DNA–Protein Noncovalent Cross‐Linking: Ruthenium Dipyridophenazine Biotin Complex for the Assembly of Proteins and Gold Nanoparticles on DNA Templates

We report the first example of a small molecule that can noncovalently cross-link DNA with streptavidin and streptavidin-labeled materials. Molecule 1 possesses a ruthenium dipyridophenazine DNA-intercalating moiety and a biotin unit; these two units are adequately separated to ensure efficient cross-linking of DNA with protein. Complex 1 is essentially nonemissive in aqueous solution and when bound to streptavidin, however, its luminescence is turned "on" when it binds to DNA. We have used these properties to establish that this complex can simultaneously bind to DNA and streptavidin, and can thus bring these two biomolecules together. We also synthesized a related molecule, 3, in which the biotin and DNA-intercalating moieties are covalently bound. While complex 3 can intercalate into DNA through a threading mechanism, luminescence experiments show that it cannot simultaneously bind DNA and streptavidin, most likely due to the proximity of its two molecular-recognition units. The cross-linking ability of molecule 1 was used to template the assembly of streptavidin molecules on circular plasmid DNA, as visualized by atomic force microscopy. In addition, using 1, we show the organization of discrete groups of gold nanoparticles labeled with streptavidin on a linear DNA template of finite size, with transmission electron microscopy. In these experiments the DNA template acted as a "molecular ruler" that dictated the number of particles in the assembly.

A Facile Route to Bimetallic Ruthenium Dipyridophenazine Complexes

Using achiral coordinatively unsaturated metal complex building-blocks, the two step synthesis of a bimetallic complex containing independent [Ru(II)dppz] units tethered together by a linking 4,4'dipyridyl-1,5-pentane ligand is reported. Photophysical studies on this prototype system indicate that the characteristic luminescence of the [Ru(II)dppz] moieties is perturbed by self-quenching processes. Preliminary binding studies on the complex with natural and synthetic duplex DNA is reported. Luminescence and calorimetric titrations reveal that the complex does not show enhanced binding affinity with respect to analogous monometallic complexes. This result is interpreted by a consideration of the length and rigidity of the linker employed in the complex.

A ruthenium dipyridophenazine complex that binds preferentially to GC sequences.

Uniquely, a Ru11 complex of the dppz ligand shows a preference for GC sequences of DNA.

Synthesis and electronic properties of conjugated polymers based on rhenium or ruthenium dipyridophenazine complexes

Two series of luminescent conjugated polymers based on rhenium(I) or ruthenium(II) dipyridophenazine complexes have been synthesized by the palladium catalyzed coupling reaction using divinylbenzene and aromatic dibromides as the monomers. From the photoluminescence spectra, an energy transfer process between the conjugated backbone and the metal complexes is proposed. The ruthenium containing polymers were fabricated into light emitting devices because of their better film forming qualities. The electroluminescence (EL) of the polymers originated from the emission due to the π*-π or π*-d transitions. Depending on the metal complex content, the polymers exhibit different absorption and EL spectra. The devices exhibit a turn-on voltage of 10 V and external quantum efficiency of 0.6%. From the cyclic voltammetry results, an additional oxidation couple was observed after the incorporation of the ruthenium complex which may contribute to the charge transport process. This was further supported by the charge mobilities measurement in which the electron and hole mobilities of the polymers are enhanced by the metal complex and are of the order of 10 –4 cm 2 V –1 s –1 .

Tuning the mechanism of DNA cleavage photosensitized by ruthenium dipyridophenazine complexes by varying the structure of the two non intercalating ligands

The influence of the nature of ligands on the efficiency of ruthenium complexes for photosensitizing DNA cleavage was investigated. Ru(bipy) 2dppz 2+ and Ru(bpz) 2dppz 2+ were selected as DNA breakers on the basis of their high affinity for DNA due to the presence of a dppz ligand which can partially intercalate in the major groove of DNA. Their photosensitizing properties were compared to those of Ru(bipy) 3 2+, a complex which binds to DNA with a far lower constant. Upon irradiation, these complexes promoted the formation of single strand breaks in supercoiled ΦX 174 DNA. Unexpectedly, Ru(bipy) 2dppz 2+ was found to be less efficient than Ru(bipy) 3 2+ whatever the dye concentration or the [base pair]/[Ru] molar ratio r. Scavenging experiments have shown that the oxidative DNA cleavage induced by Ru(bipy) 2dppz 2+ mainly results from a Type II mechanism. The behavior of Ru(bipy) 2dppz 2+ was different: this compound was clearly more efficient than Ru(bipy) 3 2+ as DNA breaker and its efficiency was not modified by the presence of oxygen or by addition of scavengers of reactive oxygen species. In this case, a mechanism involving electron transfer between the excited state of the ruthenium complex and the guanine residue was proposed in agreement with emission lifetime measurements. The change in mechanism observed between Ru(bipy) 2dppz 2+ and Ru (bipy) 2dppz 2+ results from an increase of the reduction potential of the ruthenium complexes in the excited state, which appears to be the main factor controlling the efficiency.