Restriction endonuclease EcoRI alters the enantiomeric preference of chiral metallointercalators for DNA: an illustration of a protein-induced DNA conformational change

A conformational change in the DNA plasmid ColE1 appears to occur upon specific binding of the restriction endonuclease EcoRI. Enzyme association alters the chiral discrimination found in binding metallointercalators to DNA sites. The complexes tris(1,10-phenanthroline)ruthenium(II), Ru(phen)3(2+), tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II), Ru(DIP)3(2+), and tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), Co(DIP)3(3+), in general, bind stereoselectively to DNA helices, with enantiomers possessing the delta configuration bound preferentially by right-handed B-DNA. In the presence of EcoRI, however, this enantioselectivity is altered. The chiral intercalators, at micromolar concentrations, inhibit the reaction of EcoRI, but for each enantiomeric pair it is the lambda enantiomer, which binds only poorly to a B-DNA helix, that inhibits EcoRI preferentially. Kinetic studies in the presence of lambda-Ru(DIP)3(2+) indicate that the enzyme inhibition occurs as a result of the lambda enantiomer binding to the enzyme-DNA complex as well as to the free enzyme. Furthermore, photolytic strand cleavage experiments using Co(DIP)3(3+) indicate that the metal complex interacts directly at the protein-bound DNA site. Increasing concentrations of bound EcoRI stimulate photoactivated cleavage of the DNA helix by lambda-Co(DIP)3(3+), until a protein concentration is reached where specific DNA recognition sites are saturated with enzyme. Thus, although lambda-Co(DIP)3(3+) does not bind closely to the DNA in the absence of enzyme, specific binding of EcoRI appears to alter the DNA structure so as to permit the close association of the lambda isomer to the DNA helix. Mapping experiments demonstrate that this association leads to photocleavage of DNA by the cobalt complex at or very close to the EcoRI recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of cellular uptake of a ruthenium polypyridyl complex

Transition metal complexes provide a promising avenue for the design of therapeutic and diagnostic agents, but the limited understanding of their cellular uptake is a roadblock to their effective application. Here, we examine the mechanism of cellular entry of a luminescent ruthenium(II) polypyridyl complex, Ru(DIP) 2dppz (2+) (where DIP = 4,7-diphenyl-1,10-phenanthroline and dppz = dipyridophenazine), into HeLa cells, with the extent of uptake measured by flow cytometry. No diminution of cellular uptake is observed under metabolic inhibition with deoxyglucose and oligomycin, indicating an energy-independent mode of entry. The presence of organic cation transporter inhibitors also does not significantly alter uptake. However, the cellular internalization of Ru(DIP) 2dppz (2+) is sensitive to the membrane potential. Uptake decreases when cells are depolarized with high potassium buffer and increases when cells are hyperpolarized with valinomycin. These results support passive diffusion of Ru(DIP) 2dppz (2+) into the cell.     

Design and simple routes of synthesis of oligonucleotide conjugates for studies of DNA triple helix formation

A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2',3'-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.     

Activation and deactivation of a carbene containing non-classical ruthenium hydride complex in catalytic processes involving C-H bond cleavage

The carbene-containing non-classical ruthenium hydride complex [(PCy₃)Ru(H)₂(H₂)₂(IMes)] 1 (IMes=1,3-Bis-(2,4,6-trimethylphenyl)imidazol-2-ylidene) is an active catalyst for H/D exchange in aromatic ketones. It is also capable of combining sp² C-H bond activation with C-C bond formation. Comparing the chemo- and regio-selectivities of the H/D exchange process and the C-C bond formation clearly indicates that different intermediates are involved in the two processes. High pressure NMR studies provide strong evidence that the key intermediate for the C-C coupling reaction is analogous to that for other ruthenium catalysts reported previously. Catalytic turnover is limited by the instability of this intermediate in the presence of the olefinic coupling partner.     

Comparing the spectroscopic and electrochemical properties of ruthenium and osmium complexes of the tridentate polyazine ligands 2,2′:6′,2″-terpyridine and 2,3,5,6-tetrakis(2-pyridyl)pyrazine

A number of osmium and ruthenium complexes of the tridentate ligands 2,2′:6′,2″-terpyridine (tpy) and 2,3,5,6-tetrakis(2-pyridyl)pyrazine (tpp) have been prepared and characterized by our laboratory. All these complexes possess metal based oxidations and ligand based reductions localized on each polyazine ligand. Polymetallic complexes bridged by the tpp ligand exhibit two sequential tpp based reductions prior to the reduction of other polyazine ligands in these complexes. The spectroscopy of these complexes is dominated by ligand based π-π^* transitions in the ultraviolet and MLCT (metal-to-ligand charge transfer) bands terminating on each polyzine ligand in the visible. For the complexes reported herein the lowest lying optical transitionis a M → BL CT band. For most of the complexes reported, occupation of this excited state gives rise to an observable emission at room temperature. For ruthenium complexes of these tridentate ligands, the presence of a low-lying LF state shortens the excited state lifetimes of these chromophores. This gives rise to ruthenium complexes that display shorter excited state lifetimes than the analogous osmium based systems. Incorporation of tpp based chromophores into polymetallic frameworks leads to the production of bimetallic species with long-lived excited states, 100 ns at room temperature. This makes these chromophores good candidates for the development of stereochemically defined supramolecular complexes. It is possible to measure an electrochemical HOMO-LUMO energy gap and a correlation between this electrochemically measured energy gap and the spectroscopic energy associated with this HOMO→LUMO transition are reported herein (HOMO== highest occupied molecular orbital, LUMO = lowest unoccupied molecular orbital).     

Catalysis of aerobic olefin oxidation by a ruthenium perhaloporphyrin complex

The perhalogenated porphyrin ruthenium complex (TFPPCl₈)Ru(CO) (TFPPCl₈ = octachlorotetrakis(pentafluorophenyl)porphyrin) catalyzes aerobic oxidation of olefins at room temperature. Cyclohexene is oxidized primarily at the allylic position, and styrene primarily to benzaldehyde, indicating a radical autoxidation mechanism. Reactions are enhanced by visible light. Reaction with _m-chloroperbenzoic acid converts the ruthenium complex to (TFPPCl₈)Ru(O)₂, but such oxo complexes do not appear to participate in catalytic aerobic oxidation.     

DESIGN AND SIMPLE ROUTES OF SYNTHESIS OF OLIGONUCLEOTIDE CONJUGATES FOR STUDIES OF DNA TRIPLE HELIX FORMATION

A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2′,3′-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.     

The dichotomy in the DNA-binding behaviour of ruthenium(II) complexes bearing benzoxazole and benzothiazole groups

The substituted tris(bipyridine)ruthenium(II) complexes {[Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) [where bpy=2,2'-bipyridine and bbob=bis(benzoxazol-2-yl)-2,2'-bipyridine] have been prepared and compared to the previously studied complex [Ru(bpy)(2)(4,4'-bbtb)](2+) [where bbtb=bis(benzothiazol-2-yl)-2,2'-bipyridine]. From the UV/VIS titration studies, Delta-[Ru(bpy)(2)(4,4'-bbob)](2+) displays a stronger association than the Lambda-isomer with calf-thymus DNA (ct-DNA). For [Ru(bpy)(2)(5,5'-bbob)](2+), there appears to be minimal interaction with ct-DNA. The results of fluorescence titration studies suggest that [Ru(bpy)(2)(4,4'-bbob)](2+) gives an increase in emission intensity with increasing ct-DNA concentrations, with an enantiopreference for the Delta isomer, confirmed by membrane dialysis studies. The fluorescent intercalation displacement studies revealed that [Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) display a preference for more open DNA structures such as bulge and hairpin sequences. While Lambda-[Ru(bpy)(2)(4,4'-bbtb)](2+) has shown the most significant affinity for all the oligonucleotides sequences screened in previous studies, it is the Delta isomer of the comparable benzoxazole ruthenium(II) complex (Delta-[Ru(bpy)(2)(4,4'-bbob)](2+)) that preferentially binds to DNA.     

In vivo photoreaction of a chiral cobalt complex: DNA cleavage by Co(DIP)3(3+) in mammalian cells

The chiral complex tris (diphenylphenanthroline) cobalt (III) (Co(DIP)3(3+) provides a photoreactive probe for chromatin structure in mammalian cells. The complex, which upon photoactivation cleaves DNA in a conformation-specific fashion in vitro, is shown also to cleave DNA in vivo upon irradiation with ultraviolet light (greater than 300 nm). delta- and lambda-Co (DIP)3(3+) isomers are taken up efficiently into cultured Chinese hamster ovary cells and concentrate within cell nuclei. In the absence of light the complexes are toxic to the cells (10% survival at approximately 300 nM), but after ultraviolet irradiation, the toxicity is markedly (greater than 10-fold) increased. The synergism between irradiation and Co(DIP)3(3+) administration may lie in photoreactions with DNA elicited by the cobalt complex. Alkaline sucrose gradient analysis of DNA from cells exposed to lambda-Co(DIP)3(3+) and irradiation show single-stranded DNA fragmentation under conditions where little cleavage is seen in cells either incubated with lambda-Co(DIP)3(3+) or irradiated with greater than 300 nm A ultraviolet light. Cellular DNA is cleaved with lower efficiency than naked DNA, likely due to decreased accessibility of sites in vivo. Hybridization of fragments obtained from the alkaline sucrose gradients to a probe specific for the amplified dihydrofolate reductase gene reveals a similar distribution of dhfr sequences and total DNA, indicating that the family of conformations recognized by lambda-Co(DIP)3(3+) are dispersed throughout the genome.     

Differences in structure, physiological stability, electrochemistry, cytotoxicity, DNA and protein binding properties between two Ru(III) complexes

A novel Ru(III) complex, mer-[RuCl(3)(CH(3)CN)(dpq)] (1), has been synthesized and characterized by X-ray diffraction, where dpq=dipyrido[3,2-d:2',3'-f]quinoxaline. Its chemical and biological properties have been intensively compared with those of mer-[RuCl(3)(DMSO)(dpq)] (DMSO=dimethyl sulfoxide) (2). It has been found that the stability in buffered solutions and the reduction potential for the Ru(III)/Ru(II) couple can be modulated by changing the small molecule bonded to the Ru(III) center. Interactions of 1 with DNA have been investigated by DNA melting experiments, DNA competitive binding with EB (ethidium bromide), plasmid DNA cleavage experiments and viscosity measurements. The interaction of 1 and 2 with BSA (bovine serum albumin) has also been studied using fluorescent quenching method. The experimental results show that 1 exerts higher affinity towards DNA and BSA than 2 does. The cytotoxicity of 1 has been evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method, and 2 shows slightly higher anticancer potency than 1 does against all the cell lines screened. Attempts are made to clarify the possible antitumor mechanisms of these two complexes by analyzing the experimental results presented.     

Synthesis,Structure, DNA‐Binding Properties,and Cytotoxicity of Ruthenium(II) Polypyridyl Complexes

Many ruthenium(II) complexes show high antitumor activities, and the in vitro antitumor activities are usually related to DNA binding. We designed and synthesized two Ru^II polypyridyl complexes, [Ru(dmp)₂(fpp)]²⁺ (dmp=2,9‐dimethyl‐1,10‐phenanthroline; fpp=2‐[3,4‐(difluoromethylenedioxy)phenyl]imidazo[4,5‐f] [1,10]phenanthroline and [Ru(phen)₂(fpp)]²⁺ (phen=1,10‐phenanthroline). The DNA‐binding properties of these complexes have been investigated by spectroscopic titration, DNA melting experiments, viscosity measurements, and photoactivated cleavage. The mechanism studies of photocleavage revealed that singlet oxygen (¹O₂) and superoxide anion radical (O$\rm{{_{2}^{{^\cdot} -}}}$ ) may play an important role in the photocleavage. The cytotoxicity of complexes 1 and 2 have been evaluated by MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide) method; complex 2 shows slightly higher anticancer potency than 1 does against all the cell lines screened.     

Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: probing partial intercalation and binding properties

The binding of [Ru(PDTA-H₂)(phen)]Cl (PDTA = propylene-1,2-diaminetetra-acetic acid; phen = 1,10 phenanthroline) with ctDNA (=calf thymus DNA) has been investigated through intrinsic and induced circular dichroism, UV-visible absorption and fluorescence spectroscopies, steady-state fluorescence, thermal denaturation technique, viscosity and electrochemical measurements. The latter indicate that the cathodic and anodic peak potentials of the ruthenium complex shift to more positive values on increasing the DNA concentration, this behavior being a direct consequence of the interaction of both the reduced and oxidized form with DNA binding. From spectrophotometric titration experiments, the equilibrium binding constant and the number of monomer units of the polymer involved in the binding of one ruthenium molecule (site size) have been quantified. The intrinsic circular dichroism (CD) spectra show an unwinding and a conformational change of the DNA helix upon interaction of the ruthenium complex. Quenching process, thermal denaturation experiments and induced circular dichroism (ICD) are consistent with a partial intercalative binding mode.     

Adeno-associated virus mediated gene transfer into primary rat brain neuronal and glial cultures: enhancement with the pH-sensitive surfactant dodecyl 2-(1'-imidazolyl) propionate

This study evaluated the effects of a novel, pH-sensitive surfactant, dodecyl 2-(1'-imidazolyl) propionate (DIP), on cationic lipid mediated transfection in primary rat brain neuronal and glial cultures. The cationic lipid complex DOTAP/DOPE (1, 2-dioleoyl-3-trimethylammonium propionate and dioleoyl phosphatidylethanolamine, respectively) was added over a range of concentrations (0-120 microg/ml) with DNA concentration kept constant (1.6 microg/ml). The neuron-specific enolase (NSE) and cytomegalovirus (CMV) promoters were found to drive green fluorescent protein (GFP) expression in neuron-enriched and glial cultures, respectively, using adeno-associated virus (AAV) derived constructs. NSE-driven GFP expression was not observed in glial cultures. Addition of DOTAP/DOPE increased transfection efficiency over a wide range of lipid concentrations (5-50 microg/ml) keeping DNA concentration constant (1.6 microg/ml). Addition of DIP to the lipid/DNA complex increased maximum transfection efficiencies in glial and neuronal cultures 2-3-fold. Transfection efficiencies were at their maximum with a similar total lipid concentration (50 microg/ml) in both cell-types in the presence of DIP. Neuronal cultures were more sensitive than glia to the toxic actions of DOTAP/DOPE, with or without DIP. These results indicate that AAV-mediated gene-transfer to neurons and glia can be facilitated by addition of a pH-sensitive surfactant to cationic liposome/DNA complexes and that endosomal escape could be a limiting factor in transgene expression.     

In vivo inhibition of E. coli growth by a Ru(II)/Pt(II) supramolecule [(tpy)RuCl(dpp)PtCl2](PF6)

Supramolecular complexes consisting of ruthenium chromophores and a cisplatin unit represent an emerging class of bioactive molecules of interest as anti-cancer agents. Although the ability of Ru(II)/Pt(II) heteronuclear complexes to bind to DNA has been demonstrated, the in vivo activity of these complexes has not yet been reported. In the present work, we report the anti-bacterial activity of the complex [(tpy)RuCl(dpp)PtCl(2)](PF(6)) (where dpp=2,3-bis(2-pyridyl)pyrazine, tpy=2,2':6',2'-terpyridine). The impact on bacterial cell growth of exposure to different concentrations of [(tpy)RuCl(dpp)PtCl(2)](PF(6)) and cisplatin was studied. The bioactivity of this complex was found to be due to the presence of the cis-PtCl(2) moiety, as the monometallic synthon [(tpy)RuCl(dpp)](PF(6)) did not inhibit bacterial cell growth.     

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.     

DNA binding of iron(II) mixed-ligand complexes containing 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline

Absorption spectroscopy and circular dichroism (CD) have been used to characterize the DNA binding of [Fe(phen)3]2+, [Fe(phen)2(DIP)]2+ and [Fe(phen)(DIP)2]2+ where phen and DIP stand for 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline, respectively. Both [Fe(phen)3]2+ and [Fe(phen)2(DIP)]2+ bind weakly to calf thymus DNA (CT-DNA) in an electrostatic mode, while [Fe(phen)(DIP)2]2+ binds more strongly to CT-DNA, possibly in an intercalation mode. The hypochromicity, red shift and Kb increase in the order [Fe(phen)3]2+ < [Fe(phen)2(DIP)]2+ < [Fe(phen)(DIP)2]2+ in accordance with the increase in size and hydrophobicity of the iron(II) complexes. The thermodynamic parameters obtained suggest that the DNA binding of both [Fe(phen)3]2+ and [Fe(phen)2(DIP)]2+ is entropically driven, while that of [Fe(phen)(DIP)2]2+ is enthalpically driven. A strong CD spectrum in the UV and visible region develops upon addition of CT-DNA into the racemate solution of each iron(II) complex (Pfeiffer effect). This has revealed that a shift in diastereomeric inversion equilibrium takes place in the solution to yield an excess of one of the DNA-complex diastereomers. The striking resemblance of the CD spectral profiles to those of the pure delta-enantiomer indicates that the delta-enantiomer of the iron(II) complexes is preferentially bound to CT-DNA. The mechanism of the development of Pfeiffer CD is proposed on the basis of kinetic studies on the DNA binding of the racemic iron(II) complexes.     

DNA-binding studies of mixed-ligand (ethylenediamine)ruthenium(II) complexes

A series of mixed-ligand ruthenium(II) complexes of the type [Ru(en)(2)bpy](2+) (bpy=2,2-bipyridine; 1), [Ru(en)(2)phen](2+) (phen=1,10-phenantroline; 2), [Ru(en)(2)IP](2+) (IP=imidazo[4,5-f][1,10]phenanthroline; 3), and [Ru(en)(2)PIP](2+) (PIP=2-phenylimidazo[4,5-f][1,10]phenanthroline; 4) have been isolated and characterized by UV/VIS, IR, and (1)H-NMR spectral methods. The binding of the complexes with calf thymus DNA has been investigated by absorption, emission spectroscopy, viscosity measurements, DNA melting, and DNA photo-cleavage. The spectroscopic studies together with viscosity measurements and DNA melting studies support that complexes 1 and 2 bind to CT DNA (=calf thymus DNA) by groove mode. Complex 2 binds more avidly to CT DNA than complex 1, complexes 3 and 4 bind to CT DNA by intercalation mode, 4 binds more avidly to CT DNA than 3. Noticeably, the four complexes have been found to be efficient photosensitisers for strand scissions in plasmid DNA.     

The use of the borocaptate anion as a ligand. Synthesis and X-ray crystal structure of pentaammine (1-thiolato-closo-undecahydrododecaborane)ruthenium(III) dihydrate

The complex [Ru(SB₁₂H₁₁)(NH₃)₅]·2H₂O has been prepared by the reaction of Cs₂B₁₂H₁₁SH with [RuCl(NH₃)₅]Cl₂ in aqueous solution. The complex represents the first reported example of the borocaptate anion acting as a ligand. The structure of the complex has been determined by single crystal X-ray diffraction analysis. The crystal parameters are monoclinic, space group P2₁/c, A = 8.056(1), B = 14.240(2), C = 15.172(2) Å, β=98.48° and Z = 4. The ruthenium atom has a distorted octahedral coordination. The distortion is probably due to the high (3⁻) charge and the large bulk of the borocaptate ligand. These features can also be observed in the spectroscopic properties of the complex.     

DNA conformational changes and cleavage by ruthenium(II) nitrofurylsemicarbazone complexes

Metal complexes that establish interactions with DNA are being studied not only because of their potential use as therapeutic agents but also as tools for biochemistry and molecular biology. Searching for drugs with anti-trypanosome activity, we previously synthesized a series of ruthenium mixed ligand dimethyl sulfoxide complexes of the type [Ru(II)Cl(2)(DMSO)(2)L], where L is 5-nitrofurylsemicarbazone derivatives and DMSO is dimethyl sulfoxide. Though they present the ability to bind DNA, no activity against parasites in cell culture was observed. Considering their potential application as molecular tools we further analyzed the interactions with DNA through an electrophoretic approach. Non covalent withdrawal of superhelicity and a rapid nicking activity upon covalent interaction was observed. Inhibition of both effects was observed in the presence of distamycin suggesting the involvement of the DNA minor groove in the interaction with the nitrofurylsemicarbazone ruthenium complexes. In addition cleavage inhibition by dimethyl sulfoxide suggests an oxidative mechanism of action.     

Photophysical properties, X-ray structures, electrochemistry, and DFT computational chemistry of osmium complexes

We report the synthesis of phosphorescent divalent osmium complexes of the form [Os(N-N)₂(L-L) or Os(L-L)₂(N-N)]²⁺ (PF₆)₂ where N-N is a derivative of 1,10-phenanthroline, and L-L is a diarsine or diphosphine ligand: 1,2-bis(dimethylphosphino)ethane, 1,2-bis(dicyclohexylphosphino)ethane, or 1,2-bis(dimethylarseno)benzene. X-ray structures have been determined, luminescent and electrochemical properties have been measured and DFT calculations have been performed on the complexes. The emission lifetime of complexes of structure Os(II)(L-L)₂(N-N) are longer than the those of Os(II)(N-N)₂(L-L). The DFT calculations show that there is significant mixing of the π−π^∗ into the dπ−π^∗ charge-transfer state for the complexes of the form Os(II)(L-L)₂(N-N) resulting in a longer lived excited state. Through DFT calculations we were able to conclude that the HOMO of the complexes is a d orbital on the osmium while the LUMO is the b₁(ψ) π^∗ system of the phenanthroline. However, we found that the HOMO did not have the correct symmetry to enable strong charge transfer to the phenanthroline to be observed, and the strong MLCT transition observed in the spectra is the metal d HOMO(−1) to the b1 π^∗ LUMO of the phenanthroline.