Aryl-Substituted Ruthenium(II) Complexes: A ... - ACS Publications

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Aryl-Substituted Ruthenium(II) Complexes: A Strategy for Enhanced Photocleavage and Efficient DNA Binding Felipe Diógenes Abreu,† Tercio de F. Paulo,† Marcelo H. Gehlen,‡ Rômulo A. Ando,§ Luiz G. F. Lopes,† Ana Cláudia S. Gondim,† Mayron A. Vasconcelos,∥,⊥ Edson H. Teixeira,∥ Eduardo Henrique Silva Sousa,*,† and Idalina Maria Moreira de Carvalho*,† †

Laboratório de Bioinorgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, Cx. Postal 6021, 60440-900 Fortaleza, Ceará Brazil ‡ Instituto de Química de São Carlos, Universidade de São Paulo, 13566-590 São Carlos, São Paulo, Brazil § Instituto de Química, Universidade de São Paulo, Cx. Postal 26077, 05508-000 São Paulo, Brazil ∥ Laboratório Integrado de Biomoléculas, Departamento de Patologia e Medicina Legal, Universidade Federal do Ceará, 60441−750 Fortaleza, Ceará Brazil ⊥ Departamento de Ciências Biológicas, Faculdade de Ciências Exatas e Naturais, Universidade do Estado do Rio Grande do Norte, 59625-620, Mossoró, Rio Grande do Norte, Brazil S Supporting Information *

ABSTRACT: Ruthenium polypyridine complexes have shown promise as agents for photodynamic therapy (PDT) and tools for molecular biology (chromophore-assisted light inactivation). To accomplish these tasks, it is important to have at least target selectivity and great reactive oxygen species (ROS) photogeneration: two properties that are not easily found in the same molecule. To prepare such new agents, we synthesized two new ruthenium complexes that combine an efficient DNA binding moiety (dppz ligand) together with naphthyl-modified (1) and anthracenylmodified (2) bipyridine as a strong ROS generator bound to a ruthenium complex. The compounds were fully characterized and their photophysical and photochemical properties investigated. Compound 2 showed one of the highest quantum yields for singlet oxygen production ever reported (ΦΔ= 0.96), along with very high DNA binding (log Kb = 6.78). Such photochemical behavior could be ascribed to the lower triplet state involving the anthracenyl-modified bipyridine, which is associated with easier oxygen quenching. In addition, the compounds exhibited moderate selectivity toward G-quadruplex DNA and binding to the minor groove of DNA, most likely driven by the pendant ligands. Interestingly, they also showed DNA photocleavage activity even upon exposure to a yellow light-emitting diode (LED). Regarding their biological activity, the compounds exhibited an exciting antibacterial action, particularly against Gram-positive bacteria, which was enhanced upon blue LED irradiation. Altogether, these results showed that our strategy succeeded in producing light-triggered DNA binding agents with pharmacological and biotechnological potential.

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quenched by molecular oxygen to generate 1O2;1 however, the low DNA binding affinity of [Ru(bpy)3]2+ makes this molecule a nonselective agent, and this has limited its further application. Ruthenium and dppz-based metallointercalators have proven to be powerful molecular light-activated switches for binding and detecting DNA but with a significant drop in ROS generation. Two classical intercalators, [Ru(bpy)2(dppz)]2+ and [Ru(phen)2(dppz)]2+ (where bpy = 2,2′-bipyridine, dppz = dipyrido [3,2a:2,3-c]phenazine, and phen = phenanthroline), have been extensively investigated by Barton and colleagues3−5 using spectroscopic techniques. For molecular recognition of specific DNA sequence (G-quad-

INTRODUCTION Photodynamic therapy (PDT) is an important strategy against cancer because it is a noninvasive intervention that uses visible light to enhance the action of a photosensitizer drug in the tumor region. Thus, PDT provides temporal and spatial selectivity for cancer treatment.1 The chemical and optical characteristics of many photosensitizers preclude their effective use in tumor-tissue treatment.2 It is therefore important to find new light-active molecules that can effectively generate reactive oxygen species (ROS), such as singlet oxygen ( 1O 2). Ruthenium(II) polypyridine complexes may be tuned to exhibit suitable optical properties (e.g., long-wavelength bands, strong absorptivity) along with high quantum yield for 1O2 formation, allowing their possible use in PDT. The triplet metal-to-ligand charge-transfer (3MLCT) state of [Ru(bpy)3]2+ can be © 2017 American Chemical Society

Received: May 2, 2017 Published: July 20, 2017 9084

DOI: 10.1021/acs.inorgchem.7b01108 Inorg. Chem. 2017, 56, 9084−9096

Article

Inorganic Chemistry

measurements were performed in a single-compartment glass cell filled with deaerated (argon-purged) acetonitrile containing a tetra-Nbutylammonium perchlorate (0.1 mol L−1) electrolyte solution and equipped with glassy-carbon working, platinum-wire auxiliary, and Ag/ AgCl pseudoreference electrodes. All of the potentials described in this study were reported versus Ag/AgCl electrodes, which, under the given experimental conditions, gave a value of 0.54 V for the ferrocene/ferrocenium couple. Materials. Acetonitrile, 2-aminoanthracene (anth), 2-aminonaphthalene (naph), tetrafluorboric acid, 1,3-diisopropilcarbodiimide, 4,4′dimethyl-2,2′-bipyridine, selenium dioxide, 1-hydroxybenzotriazole hydrate, N-methylmorpholine, ethidium bromide (EB), Hoechst 33258, methyl green, ruthenium(III) chloride hydrate, and ruthenium trisbipyridine chloride from Aldrich Co, methanol from Mallinckrodt, and sulfuric acid and trifluoracetic acid from Merck were all used as received. Acetone (Mallinckrodt) was treated with sodium sulfate and then distilled and stored over 4 Å molecular sieves. Dimethylformamide (DMF; Merck) was distilled under reduced pressure at 75 °C, kept dry, and stored over 4 Å molecular sieves. Diethyl ether (Synth) was treated with metallic sodium and then distilled twice before use. Calf-thymus DNA (CT-DNA; stored at 4 °C) was purchased from Sigma-Aldrich. DNA oligomers 5′-G3(T2AG3)3- 3′ (HTG21), the complementary cytosine-rich strand 5′-C3(TA2C3)3-3′(ssDNA), the CC mismatch ssDNA 5′-ATCACACCGAACACTCC-3′, and the complementary strand 3′-TAGTGTGCCTTGTGAGG-5′ were purchased from IDT, Inc. Single-stranded extinction coefficients were calculated from mononucleotide data using a nearest-neighbor approximation. The preparations of G-quadruplex, single-stranded, matched, and mismatched DNA were carried out as follows: the oligonucleotide samples, dissolved in different buffers, were mixed when necessary (matched and mismatched DNA), heated to 90 °C for 5 min, spontaneously cooled to room temperature, and then incubated at 4 °C overnight. Buffer A: 10 mmol L−1 Tris-HCl, pH 7.4. Buffer B: 10 mmol L−1 Tris-HCl, 100 mmol L−1 KCl, pH 7.4. Stock solutions were stored at 4 °C and used after no more than 4 days. Further dilution was made in the corresponding buffer to the required concentrations for all of the experiments. Syntheses. 4′-Methyl-2,2′-bipyridine-4-carboxylic acid (mbpyOH),13 4′-methyl-N-(anthracen-2-yl)-2,2′-bipyridine-4-carboxyamide (mbpy-anth), and 4′-methyl-N-(naphthalen-2-yl)-2,2′-bipyridine-4carboxyamide (mbpy-naph),14 and dipyrido[3,2a:2,3-c]phenazine (dppz)9 were prepared following the procedure described in the literature. [Ru(bpy)(dppz)(L)](PF6)2 Complexes. The precursor complex cis[Ru(bpy)(dppz)Cl2]·3H2O was synthesized according to the literature procedure.9,15 Then, [Ru(bpy)(dppz)(L)](PF6)2 complexes were prepared as follows: 100 mg (0.163 mmol) of cis-[Ru(bpy)(dppz)Cl2] and 0.163 mmol of L (mbpy-anth or mbpy-naph) were dissolved in 30 mL of a solution of water/ethanol (1:1) and kept under stirring and refluxed for 8 h. This mixture was concentrated by rotary evaporation and loaded onto a silica gel column, which was eluted with DMF/ methanol (1:1). An orange running band was collected containing the desired complex, which was dried by rotary evaporation, dissolved in a minimum volume of DMF, and precipitated by adding a few drops of concentrated NH4PF6, followed by the addition of anhydrous diethyl ether. The precipitate was filtered off, washed with an excess of anhydrous ether, and dried under vacuum. Spectroscopic data for the mixture of geometric isomers of 1. 1H NMR [Figure S1; (CD3)2SO]: δ 10.94−10.85 (2H, NH), 9.69−9.61 (4H, H2dppz and H11dppz), 9.30− 9.24 (2H, H3a), 8.97−8.83 (4H, H5′bpy and H4′bpy), 8.58−8.50 (4H, H5dppz and H8dppz), 8.45−8.37 (2H, H3′a), 8.36−8.31 (2H, H6a), 8.29−8.17 (12H, H4dppz, H9dppz, H6′bpy, H3′bpy, H6dppz, and H7dppz), 8.16−8.11 (2H, H5a), 8.08−8.01 (4H, H3dppz and H10dppz), 8.01− 7.95 (4H, H4bpy, H1b), 7.95−7.88 (2H, H3bpy), 7.88−7.82 (4H, H3b and H4b), 7.82−7.75 (2H, H8b), 7.75−7.71 (2H, H6′a), 7.67−7.60 (2H, H5b), 7.58−7.55 (2H, H5′a), 7.55−7.44 (4H, H7b and H6b), 7.55−7.37 (2H, H5bpy), 7.32−7.26 (2H, H6bpy), 2.61 and 2.53 (6H,CH3). IR: ν(CO) amide 1667 cm−1 Anal. Calcd for C50H45F12N9OP2Ru·3H2O: C, 49.76; H, 3.42; N, 10.44. Found: C, 49.72; H, 3.39; N 10.41. Spectroscopic data for the mixture of

ruplexes, B-DNA, Z-DNA, mismatches, CC- and GC-rich sequences, etc.), the metallointercalators, in general, present a well-defined symmetry and rigid structure.5 In order to enhance DNA binding and selectivity to certain DNA sequences, e.g., Gquadruplexes, B-DNA, mismatches, CC- and GC-rich sequences, etc., the shape and size of the ancillary ligands can be important and should be exploited.5−11 Chromophore-assisted light inactivation (CALI) is another useful strategy to study biological systems, and it has employed organic dyes and ruthenium(II) tris(bipyridine) complexes as ROS generators. This tool requires linkage of the ruthenium(II) complex to an antibody to provide selectivity, enabling interaction with a specific protein that will be inactivated upon light generation of 1 O2.10 In this study of visible-light-induced DNA photodamage, we have explored the influence of the ancillary ligands on DNA binding and ROS generation. For this purpose, we synthesized and characterized two RuIIdppz-based complexes with naphthyl and anthracenyl aromatic groups appended to the bipyridine ancillary ligand. The structures of complexes 1 and 2 are shown in Figure 1.

Figure 1. Chemical structures of ruthenium aryl complexes.

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EXPERIMENTAL SECTION

Physical Measurements. Absorption spectra of dilute solutions (5 × 10−5 mol L−1) were measured with a Hewlett-Packard model 8453 spectrophotometer, and the steady-state emission and excitation spectra were recorded using a PTI Quanta-Master QM-40 fluorescence spectrophotometer. Emission quantum yields were calculated relative to [Ru(bpy)3]2+ in methanol (Φem = 0.045). Luminescence decays were recorded with a laser spectrometer by timecorrelated single-photon counting using a TC900 counting board from Edinburgh Instruments as described elsewhere.12 Lifetimes were evaluated by the reconvolution procedure using FAST software from Edinburgh Instruments. Kinetic analyses of the decays were fit to biexponential decay functions: I(t) = I0[a1 exp(−t/τ1) + a2 exp(−t/τ2), where I(t) is the signal intensity as a function of time, I0 is the intensity at long time, an is a preexponential factor that represents the relative contribution from the nth component to the initial emission intensity, and τn is the lifetime of the nth component. All solutions were deoxygenated with argon for at least 10 min prior to measurements. 1 H NMR spectra were obtained in the designated solvents on a Bruker (300 MHz) spectrometer. Electrochemical data were obtained by cyclic voltammetry using an Epsilon potentiostat [Bioanalytical Systems Inc. (BAS), West Lafayette, IN] at 25 ± 0.2 °C. The 9085


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Inorganic Chemistry Table 1. Spectroscopic and Electrochemical Properties of Complexes 1 and 2a E, V vs Ag/AgCl λabs, nm (ε × 104, mol L−1 cm−1)

complex 1 2 3 4 a

227 257 284 285

E1/2(RuIII/II)

(31), 281 (42), 360 (1.75), 458 (2.2) (52), 276 (54), 360 (1.8), 460 (2.7) (10.4), 320 (2.14), 359 (1.75), 370 (1.72), 445 (1.63)b (8.71), 323 (sh), 345 (sh), 452 (1.45)c

1.35 1.33 1.29c 1.31d

Ox 1.30

Red1

Red2

Red3

Red4

−0.71 −0.96 −0.98c −1.27d

−0.97 −1.61 −1.40c −1.46d

−1.48 −1.78 −1.63c −1.73d

−1.72

Recorded in acetonitrile at 298 K. bFrom ref 27 in water. cFrom ref 28. dFrom ref 29.

geometric isomers of 2. 1H NMR [Figure S2; (CD3)2SO]: 11.00− 10.94 (2H, NH), 9.70−9.63 (4H, H2dppz and H11dppz), 9.29−9.27 (2H, H3a), 8.98−8.85 (4H, H4′bpy and H5′bpy), 8.69−8.65 (2H, H6a), 8.63−8.59 (2H, H5a), 8.58−8.51 (4H, H5dppz and H8dppz), 8.36−8.32 (2H, H3′a), 8.28−8.23 (4H, H4dppz and H9dppz), 8.23−8.18 (4H, H6dppz and H7dppz), 8.18−8.13 (4H, H6′bpy and H3′bpy), 8.12−8.01 (4H, H3dppz and H10dppz), 8.00−7.97 (2H, H4b), 7.96−7.91 (4H, H1b and H3b), 7.88−7.83 (2H, H6′a), 7.81−7.75 (4H, H6b and H9b), 7.72−7.68 (2H, H5′a), 7.67−7.60 (4H, H7b and H8b), 7.55−7.46 (4H, H5b and H10b), 7.43−7.37 (2H, H5bpy), 7.31−7.27 (2H, H6bpy), 2.61−2.54 (6H, CH3). IR: ν(CO) amide 1668 cm−1. Anal. Calcd for C54H37F12N9OP2Ru·3H2O: C, 51.68; H, 3.29; N, 10.05. Found: C, 51.55; H, 3.32; N, 9.93. Measurement of 1O2 Photogeneration. 1,3-Diphenylisobenzofuran (DPBF) was chosen to measure the quantum yield of 1O2 generation by the ruthenium complexes. DPBF is fluorescent and reacts selectively with 1O2 to produce a nonluminescent product. A series of 2 mL air-saturated ethanol solutions containing DPBF (20 μmol L−1) and the complexes were prepared in a 1 cm path length fluorescence cuvette and illuminated with LED light of 463 nm (Basetech Conrand, 1.7 W) for 100 s. The consumption of DPBF by reaction with 1O2 was followed by monitoring of the fluorescence intensity decrease at the emission maximum (λex = 405 nm; λmax em = 479 nm) at different irradiation times. The quantum yield of the triplet state (ΦΔ) for 1O2 generation by the [Ru(bpy)3]2+ complex in airsaturated ethanol, ΦΔ = 0.84, was taken as a reference.16 DNA Binding and Photocleavage Studies. DNA titrations were followed by electronic absorption spectroscopy. A constant concentration of the ruthenium complexes (10 μmol L−1, respectively) was mixed with varying DNA concentrations (0−40 μmol L−1 in nucleotide base pairs) in 10 mmol L−1 of Tris buffer (pH 7.4). After each addition of DNA into the solution containing the ruthenium complex, the mixture was allowed to equilibrate at 25 °C for 5 min, after which the absorption and emission readings were recorded. Binding to small oligonucleotide sequences was used to investigate interactions with G-DNA quadruplex (HTG21), single-stranded DNA (ssDNA), double-stranded (dsDNA), and mismatched doublestranded DNA (CCmis). These oligos were prepared as described before and their concentrations measured by their absorbance at 260 nm. Binding studies were conducted by titrating 5 μmol L−1 DNA with complex 1 or 2 in 10 mmol L−1 Tris-HCl buffer (pH 7.4) at 22 °C and following the fluorescence emission. Free complex 1 or 2 was also titrated without DNA as a control for the basal fluorescence signal. The total volume added during titration did not exceed 1% of the initial volume of the mixture in the cuvette, and so there was no requirement for dilution correction. All of the data were collected at least in duplicate and fit to a single binding equation using Prism software with R2 at least above 0.97. Investigations on the intercalation and type of groove binding mode were done using EB, methyl green, and Hoechst agents, versus the metal complexes as competitors, in 10 mmol L−1 Tris-HCl (pH 7.4) at 22 °C. These measurements used Calf thymus DNA (CT) at 10 μmol L−1 along with 5 μmol L−1 of EB, methyl green, or Hoechst, which were titrated with metal complex 1 or 2 and monitored by fluorescence spectroscopy. All of the data were collected at least in duplicate and fit to a single binding equation using Prism software, and an apparent dissociation constant (appKd1) was obtained. The appKd1 values for metal complexes 1 and 2 were used to estimate Kd (Kd1)

using eq 1, considering a simple competition, where L is the concentration of the DNA binding agent (EB, methyl green, or Hoechst) and KdL is their dissociation constant, as reported in the literature. Controls without DNA were treated as described above to evaluate any quenching event with EB, methyl green, and Hoechst.

K d1 = Kd1(1 + L /Kd L)

app

(1)

Gel electrophoresis was done in triacetate ethylenediaminetetraacetate (TAE) buffer for the investigations of DNA photocleavage. These experiments used supercoiled pBR322 DNA (21 μmol L−1 in a nucleotide base pair) in 10 mmol L−1 Tris-HCl buffer (pH 7.4), which was mixed with ruthenium complex and irradiated or incubated in the dark. The samples were analyzed after electrophoresis in agarose gels (0.8% w/v in TAE buffer, pH 8.0) stained with EB (1 μg mL−1, 1 h), and the data were documented using a Gel Doc XR+ System (Biorad). Irradiation experiments were carried out using LED Basetech Conrand, 1.7 W (λmax em = 463, 520, and 592 nm). Computational Studies. All DFT calculations in this study were performed using the Gaussian 09 program package, revision A.02 (Gaussian Inc., Wallingford, CT).17 The geometries of complexes 1 and 2 were optimized at the density functional theory (DFT) level by means of the B3LYP functional.18−20 The 6-31G(d) basis set was used for C, H, O, and N atoms, and the LANL2DZ relativistic effective core potential basis set was used for the Ru atom. Optimized geometries in a minimum of the potential energy were confirmed by the absence of any imaginary frequency in vibrational analysis calculations. On the basis of the optimized geometries, time-dependent DFT (TD-DFT) was applied to investigate the electronic properties of the complexes and simulate the absorption spectra. TD-DFT calculations were carried out in acetonitrile solvent fields by means of the polarizable continuum model.21 The molecular orbital composition, UV−vis spectra, and assignment of principal transitions were extracted using the Multiwf n22 and GaussSum 3.023 programs, respectively. Antibacterial Activity. Microorganisms and Culture Conditions. The bacteria used in this study included Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Pseudomonas aeruginosa ATCC 10145, and Escherichia coli ATCC 11303. These strains were stored in Tryptic Soy Broth (TSB) with 20% (v/v) glycerol at −80 °C. They were inoculated in Tryptic Soy Agar (TSA) plates and incubated aerobically at 37 °C for 24 h. After growth on agar plates, individual colonies were removed and inoculated in 10 mL of a fresh TSB medium and incubated for 24 h at 37 °C under steady agitation. Prior to each antibacterial assay, the final cell concentration was adjusted to 1 × 106 colony-forming unit (cfu) mL−1. Antibacterial Assay. The susceptibility of the bacteria to ruthenium complexes was evaluated by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the complexes. The MIC values were determined according to the National Committee for Clinical Laboratory Standards M7-A6 (NCCLS, 2003), with some modifications. Briefly, the compounds were diluted in ultrapure sterile water at concentrations ranging from 3.9 to 500 μg mL−1 and dispensed into 96-well plates, with each bacterial suspension (1 × 106 cfu mL−1) in TSB. These plates were submitted to irradiation with blue LED for 1 h or kept in the dark and then incubated overnight at 37 °C. The MIC was determined to be the lowest compound concentration that completely inhibited visible bacterial growth. For the measurement of MBC, 10 μL was removed from each well, where no visible bacterial growth was observed, plated 9086


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Inorganic Chemistry onto TSA plates, and incubated at 37 °C. The MBC was considered to be the lowest antibacterial concentration of the compound for which colony growth was not observed.

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RESULTS AND DISCUSSION Characterization of Ruthenium Complexes. Spectroscopic and electrochemical studies were carried out to characterize two new complexes, 1 and 2, which were compared to [Ru(bpy)2dppz]2+ (3) and [Ru(bpy)3]2+ (4) complexes. The cyclic voltamograms for complexes 1 and 2 measured in an acetonitrile electrolyte are illustrated in Figure S3, and the data are summarized in Table 1. The half-wave potentials (E1/2) assigned to the RuIII/II redox pair of 1 and 2 were determined as 1.35 and 1.33 V versus Ag/AgCl, respectively; these were consistent with other ruthenium(II) polypyridine complexes.9,11,24 However, complex 2 presented an additional oxidation process at ca. 1.30 V. A similar process was assigned to anthracene oxidation by Padilla et al.25 Regarding the reduction processes, complexes 1 and 2 exhibited the first processes at −0.71 and −0.96 V, respectively. For complex 2, the potential is close to the first reduction potential of complex 3,11 and this suggests that the reduction process mainly involves the dppz ligand, which is more easily reduced than bpy.9 On the other hand, the first reduction process of 1 was significantly less negative. Only the second reduction process of 1 (−0.97 V) was indeed very close to the reduction potential of dppz found in 2. So, on the basis of these redox potentials, we can suggest that the lowest unoccupied molecular orbital (LUMO) of 1 resides most likely in the naphthalene moiety, while the LUMO of 2 resides on the dppz ligand.9,11,26 The other reduction potentials observed for both complexes were assigned to bipyridine reduction processes, along with processes involving reduction of the naphthalene (naph) and anthracene (anth) moieties. To study the electronic properties of 1 and 2, DFT calculations were carried out. According to the DFT results, illustrated in Figures S4−S6, the highest occupied molecular orbitals (HOMOs) of 1 and 2 were located in π(naph), with the d(Ru) contribution and anth moiety, respectively. The LUMOs of 1 and 2 resided in the dppz and bpy-naph ligands, respectively. As can be ascertained from these results, DFT predictions corroborated with the aforementioned electrochemical discussion, supporting the first reduction process being on dppz for 2 and on bpy-naph for 1. The electronic absorption spectra for the complexes in acetonitrile (Figure 2) showed high-energy transition bands between 200 and 350 nm, which correspond to π → π* transitions within the bpy and dppz ligands and a broad band around 460 nm associated with metal-to-ligand charge-transfer (MLCT). The maximum MLCT band of 3 (λmax= 440 nm)16 was blue-shifted compared to complexes 1 and 2, likely because of the lack of inductive effects from functionalized bipyridines. The spectroscopic data are shown in Table 1, which also includes data for complexes 3 and 4. Electronic transitions for the complexes were investigated using the time-dependent DFT (TD-DFT) approach. TD-DFT results suggested the contribution of singlet metal-to-ligand charge-transfer ( 1 MLCT) and metal−ligand-to-ligand charge-transfer (1MLLCT) transitions in the high-energy bands between 200 and 350 nm, besides π → π* transitions [intraligand (IL) and ligand-to-ligand charge-transfer (LLCT)] within the pendant aromatic ligands. Additionally, these calculations reinforced the finding that the low-energy absorption resulted mainly from an

Figure 2. Absorption (solid line) and emission (dashed line) spectra in acetonitrile (5 × 10−5 mol L−1) at 293 K of () 1 and (red ) 2 under excitation at 460 nm.

MLCT. The calculated spectra, excitation wavelengths, and oscillator strengths for selected transitions are presented in Figure S7 and Table S1, respectively. It is known that compound 3 exhibits a so-called light-switch effect attributed to the occupation of two separated excited states, a luminescent “bright” state (BS) and a nonluminescent “dark” state (DS), depending on the local solvent environment.16,30,31 The BS is stabilized in aprotic solvents, whereas the DS dominates in protic solvents. It was proposed that the lowest-energy emissive state for this compound arises from 3 MLCT transition from ruthenium(II) to the phenanthroline moiety of the dppz ligand, whereas the nonemissive state is localized on the phenazine moiety of the same ligand.1,3,16,30,31 There might be another low-lying electronic state for complexes 1 and 2 that has a role in the photophysical behavior, the 3MLCT state localized on the modified bipyridine with the amide group. An energy stabilization of about 0.5−2 kcal mol−1 was observed for ruthenium tris(bipyridine) complexes linked to anth and naph by an amide bridge.32 For complex 1, a weak luminescence was observed in water, while complex 2 remained completely nonluminescent. Otherwise, in acetonitrile both complexes showed emission with a maximum at 645 nm (Figure 2) and exhibited similar luminescence lifetimes (τ = 229 ns for 1 and τ = 225 ns for 2). The emission quantum yields, however, were 0.040 and 0.006 for 1 and 2, respectively. These data indicate that the emission of 1 and 2 in an aprotic solvent is from a 3MLCT state that is photophysically similar to that of the [Ru(dcbpy)2(mbpy-naph)]2+ and [Ru(dcbpy)2(mbpy-anth)]2+ as reported previously.14 Thus, the relaxation to the ground state occurs from the 3MLCT state localized on the bpy modified with the naph or anth moieties. The lower quantum yield observed for 2 was assigned to the luminescence quenching of the ruthenium 3MLCT because of the low-lying ππ* triplet state of the anth ligand (1600 cm−1 below ruthenium 3MLCT).33,34 Otherwise, for 1 (Φem = 0.040), the energy transfer from 3MLCT to the ππ* triplet state of the naph ligand was not observed because of the higher energy of this state. The emission quenching observed in a water solution is due to the DS localized on the phenazine moiety that is stabilized relative to the MLCT state corresponding to the charge-transfer state from the RuII ion 9087


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such as EB and Hoescht were also consistent with those values, as described below. The higher binding constant is probably due to an increased π density from ancillary-functionalized ligands, which contribute to the interaction with DNA. DNA Binding in Competition with EB. EB is a well-known DNA intercalator that has been used as a competitor probe to investigate new DNA intercalating agents. A maximum fluorescence emission for EB is achieved upon binding to DNA, which should exhibit its minimum upon complete displacement of the EB−DNA adduct. This lower emission should, in principle, be identical with a sample of EB without DNA, validating complete displacement. To evaluate the displacement of EB from the EB−DNA adduct, we carried out titrations of the metal complexes 1 and 2 in a mixture containing 3 μmol L−1 of EB and 10 μmol L−1 of CT-DNA in 10 mmol L−1 Tris buffer (pH 7.4), which were monitored by fluorescence. A decrease in the EB emission was observed during the addition of complex 1 or 2, supporting the theory that a competition process was taking place (Figure S8). These data were analyzed and fit to a single binding curve (R2 > 0.98), where the apparent dissociation constant (appKd) was obtained. Additionally, a competition equation (eq 1 in the Experimental Section) was used to estimate the actual Kd for DNA binding of both complexes, using a Kd value of 0.1 μmol L−1 for EB (Kb = 1 × 107).39 Interestingly, the Kd values for 1 and 2, obtained using eq 1, were 0.106 and 0.177 μmol L−1, respectively, which were reasonably close to the binding constants measured by UV−vis as reported above [UV−vis Kd of 0.146 μmol L−1 (1) and 0.166 μmol L−1 (2)]. Nevertheless, caution must be exercised in analyzing those values because complexes 1 and 2, upon binding to DNA, also emit close to the EB maximum emission band. Thus, a decrease in EB due to displacement did not lead to a full emission drop because the complexes themselves also contribute to the emission. However, these measurements supported that DNA binding of these complexes follows an intercalative mode. Effect of DNA on the Emission Lifetime. The excited-state lifetimes of 1 and 2 were determined with CT-DNA. Although only a weak luminescence was observed in water for complex 1, upon the addition of CT-DNA, the luminescence intensity increased. Complex 2 was nonluminescent in water like 3 but also presented emission after CT-DNA addition1,16 (Figure

to the bpy-L ligand. A diagram depicting these states is shown in Figure 3.

Figure 3. Schematic energy diagram of excited states of 1 and 2: GS, ground state; BS, bright state; DS, dark state.

DNA Binding Measurements. DNA Titration Monitored by Electronic Absorption Spectroscopy. DNA binding constants (Kb) were estimated by the changes in the UV−vis absorption spectra promoted by DNA addition to the ruthenium complexes (Figure 4). Indeed, during a gradual addition of CT-DNA, the MLCT band at 460 nm and the IL band at 280 nm became red-shifted to 473 and 290 nm, respectively; in addition, a decrease in their intensities was observed. The hypochromism, H (%), for the MLCT bands of the complexes varied 23% for 1 and 20% for 2. Equation 2 was used to obtain the binding affinity of the metal complexes for DNA (Figure 5a,b, insets).35 (εa − εf )/(εb − εf ) = [b − (b2 − 2Kb 2[Ru][DNA]/s)1/2 ]/2Kb[Ru]

(2)

where b = 1 + Kb[Ru] + Kb[DNA]/2s, εa, εf, and εb are the apparent, free, and bound metal complex extinction coefficients, respectively, and s is the binding size. The intrinsic binding constants were calculated as 6.83 (±0.3) × 106 L mol−1 (s = 1.1) and 6.04 (±0.2) × 106 L mol−1 (s = 0.8) for 1 and 2, respectively. These values are typical for DNA intercalation processes and agree well with the classic intercalator 3 (Kb = 3.2 × 106 L mol−1).36 It is worth noting that the DNA binding constants for both complexes are slightly higher than those for 3 (Kb = 3.2 × 106 L mol−1) and [Ru(phen)2(dppz)]2+ (Kb = 5.1 × 106 L mol−1).37,38 Other measurements using competitors

Figure 4. DNA titration monitored by UV−vis absorption changes of 1 (a) and 2 (b) in the presence of an increasing amount of salmon DNA (0− 35 μmol L−1). [Ru] = 10 μmol L−1. Inset: Plot of (εa − εf)/ (εb − εf) versus [DNA]. 9088


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Figure 5. Time-resolved luminescence of 1 (a) and 2 (b) in the presence of CT-DNA in 50 mM Tris-HCl (pH 7.4). [Ru] = 12 μmol L−1 and CTDNA = 200 μmol L−1 in base pairs.

Figure 6. Binding measurements of complexes 1 and 2 to oligonucleotides (single-stranded, ss-DNA; double-stranded, ds-DNA; single-mismatch double-stranded, ds_mis-DNA; G-quadruplex, G4-DNA) monitored by luminescence. (A) Maximum emission observed during titration of complexes 1 and 2 into oligonucleotides. (B) Measured dissociation constants for binding of complexes 1 and 2 to oligonucleotides.

measurements, we also included 100 mmol L−1 KCl to induce its formation. Interestingly, complex 1 showed a measurable luminescence even without DNA, as mentioned earlier, which is in contrast to other dppz-bound ruthenium complexes often described as light switches.1,24 On the other hand, complex 2 showed no emission at all in the absence of DNA but showed a strong emission upon DNA addition. For both compounds, however, the relative emission was significantly enhanced in the presence of the oligonucleotide sequences studied. In all cases, compared to complex 2, complex 1 exhibited better emission upon binding to DNA (Figure 6). Both complexes showed the following trend regarding the maximum emission upon DNA binding: dsDNA ∼ ds_misDNA > ssDNA ∼ G4, where binding to double-stranded DNA led to the strongest emission, and 1 emitted ca. 5-fold higher than 2. The strongest emission was quite similar for matched and mismatched sequences, suggesting that the complexes cannot recognize single-stranded DNA mistakes. It is important to note that maximum emission cannot be directly used as a criterion for a stronger thermodynamic stability, which was clearly observed below during binding studies (Figure 6). All titrations carried out were analyzed using a single binding equation, and the dissociation constants are reported (Figure S10 and Table 2). Interestingly, complexes 1 and 2 showed not much difference in the Kd values for binding to these DNA structures, as would have been expected from their differences in the maximum emission (Figure S10 and Table 2). In particular, for the G-quadruplex (G4-DNA), 1 and 2 showed the strongest binding with Kd values of 6.0 (±0.3) and 2.8 (±0.1) μmol L−1, respectively. Interestingly, as revealed by these dissociation constants, complex 2 binds 2.2-fold better to

S9). Both complexes also exhibited biexponential decay (Figure 5). The measured decay time components and respective normalized contributions were 18.3 ns (0.57) and 426 ns (0.43) for 1 and 6.5 ns (0.86) and 160 ns (0.14) for 2. These results were indicative that intercalation was the predominant binding mode with CT-DNA. This could be due to rigidity in the system upon binding to DNA, thus increasing the luminescence average lifetime of the complexes. The longer decay component may be ascribed to the intercalated ruthenium complex stabilized in the DNA (head-on mode),40−42 and the short component (side-on mode) could be related to relaxation of the prompt MLCT excited state formed upon excitation and its quenching by water or photoinduced charge migration to the DNA. Binding to G-Quadruplex, Double-Stranded, and SingleStranded DNA Structures. The interaction of the ruthenium complexes with distinct DNA structures was also investigated via emission spectroscopy using small oligonucleotides. A human telomeric single sequence (HTG21) can generate a Gquadruplex (G4) structure with K+, and this was chosen for this investigation. There is great interest in investigating Gquadruplex DNA because of its widespread presence in the human genome and its importance in gene regulation.43 A major effort has been conducted to identify selective Gquadruplex binding molecules for use in detection and therapy.44,45 Besides the G-quadruplex DNA, we also investigated how well the metal complexes would bind to single-stranded (ssDNA), double-stranded (dsDNA) DNA and also to single mismatches in double-stranded DNA (ds_misDNA). All of these measurements were carried out using 5 μmol L−1 DNA in 10 mmol L−1 Tris buffer (pH 7.4), which were titrated with complex 1 or 2. For G-quadruplex DNA 9089


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The groove binding preference can be exploited using NaI as an anionic luminescence quencher for ruthenium complexes, as described elsewhere.4 Iodide can quench the ruthenium complex bound to the DNA, providing information about the degree of protection caused by DNA polyanion regions. BDNA shows quite distinct regions, where the major groove is more accessible than the minor groove. This makes us expect a more efficient quenching process by I− if the complex binds to the major groove. Nonquenching KCl salt was also added, along with our complexes, to maintain a constant ionic strength and also evaluate ionic effects. Interestingly, no significant luminescence change was caused by either KCl or NaI (Figure S11). This behavior was further validated using 3 as a control, where there was also no significant luminescent change. Remarkably, this behavior was different for 3 as reported elsewhere, which might be due to the different DNA employed. Lim et al. reported this type of study for 3 using small oligonucleotide sequences.4 It is not unusual to find reports where quite distinct binding was noticed for ruthenium complexes depending on the DNA sequence. Besides that, a greater number of accessible sites for binding and distinct sequences in a larger DNA might lead to a more reliable study of groove interactions than a shorter sequence. Currently, there is a well-established understanding that 3 binds to the minor groove.46 This behavior has been supported by solved X-ray structures.47−49 Our results here also suggest that 3 binds to the minor groove of DNA, where a lower accessibility would prevent major quenching. This interpretation can also be drawn for complexes 1 and 2, suggesting that they interact with DNA also via the minor groove (Figure S11). This result indicated that the aryl pendent complexes might not alter the binding modes to DNA. To validate this binding mode further, we carried out a competition experiment using methyl green and Hoechst as two selective major and minor groove binders, respectively.50,51 The luminescence intensity of Hoechst bound to CT-DNA decreased significantly upon the addition of 1 and 2 (Figure 7), indicating the preference of both complexes for the minor groove, in agreement with the previous iodide study. The apparent Kd values calculated for complexes 1 and 2 during competition with Hoechst were 1.2 and 2.1 μmol L−1,

Table 2. DNA Binding Measurements for Complexes 1 and 2 Kd (μmol L−1) DNA structure

complex 1

complex 2

G-quadruplex single-stranded double-stranded single-mismatch double-stranded calf-thymus

6.0 12.9 46.1 31.4 0.146a 0.106b 0.050c

2.8 10.7 13.0 15.5 0.166a 0.177b 0.095c

a

UV−vis measurement. bLuminescence measurement in competition with EB. cLuminescence measurement in competition with Hoechst. All measurements were repeated at least twice.

G4-DNA than complex 1. This trend was also noticed for double-stranded DNA (3.5-fold) and single-mismatch doublestranded DNA binding to 2 (2.0-fold), whereas for binding of the complexes to single-stranded DNA, the Kd values were identical (Table 2). Complex 1 bound 7.7-fold stronger to Gquadruplex than to double-stranded DNA, despite having a 5fold lower maximum emission upon binding. There was an overall better binding of these DNA structures to complex 2. Because the structural difference between 1 and 2 is due to one additional aromatic ring in compound 2, these bindings may be a consequence of stronger π-stacking and/or favorable steric orientations. In summary, this quantitative study further supports that a complex can strongly interact with a DNA sequence but not necessarily reveal itself as an efficient luminescent probe. These results also support the theory that there is an exciting potential application in cancer therapy for these compounds based on their efficient interaction with G-quadruplex DNA, which deservers further investigation. Groove Binder Investigations. Because polypyridine ruthenium complexes can usually bind to DNA, either through intercalation, groove binding, or a combination of these modes,4,5 we have investigated this behavior for complexes 1 and 2 using sodium iodide and two selective minor and major groove binder compounds, Hoechst and methyl green.

Figure 7. Minor groove binder Hoechst in competition with complexes 1 (A) and 2 (B) to CT-DNA. Inset: Titration curves of complexes in the presence of Hoechst. 9090


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irradiation time were linear for all photosensitizers tested (I0 and It are the fluorescence intensities of DPBF before and after irradiation, respectively). For comparative purposes, we measured ΦΔ of the classic intercalator 3 under the same conditions. The 1O2 generation quantum yield of the photosensitizers investigated was determined according to eq 3:52 κ ΦΔ = Φs κs (3)

respectively. By using eq 1 and Hoechst binding to CT-DNA (Kd of 0.140 μmol L−1), we estimated Kd values of 0.050 and 0.095 μmol L−1, suggesting that 1 competes slightly better than 2 against Hoechst for DNA. This result supported other measurements of binding to CT-DNA, where complex 1 was slightly better than 2. As an additional control, 3 was also employed in this experiment, where it was observed to compete well against Hoechst for binding to DNA with an estimated Kd value of 0.72 μmol L−1. This behavior supported the finding that the unmodified ruthenium complex also bound mainly through the minor groove, as reported in the literature.4,5,42 All attempts to displace methyl green with complex 1 or 2 were unfruitful, with a minimum decrease in the fluorescence signal during titration, implying that no binding occurs in the major groove (Figure S12). These results indicate that the aryl pendent complexes also bind to the minor groove of DNA and both complexes are stronger binders than 3. 1 O2 Generation. The quantum yields for 1O2 generation (ΦΔ) by the ruthenium complexes were measured from the reaction of 1O2 with DPBF, a highly efficient 1O2 scavenger.52,53 The consumption of DPBF in an ethanol solution was monitored by its emission intensity decrease at 460 nm. As shown in Figure 8, plots of ln(It/I0) as a function of the

where κ is the slope of first-order plots, κs is related to the standard sensitizer (4), and Φs is its 1O2 quantum yield. The values of ΦΔ was calculated using the linear regression data from Figure 8 and eq 2, employing 4 as the standard (ΦΔ = 0.84).16 The 1O2 quantum yields were 0.96 for 2 and 0.66 for 1, which were much higher than the value observed for 3 (0.29). Thus, the pendant moieties, especially anth, provided an efficient channel to generate 1O2 via excitation at 405 nm (blue LED). This result implied potential applications in 1O2generation processes, such as photocleavage of DNA. The better result shown for 2 was assigned to the fact that its excited state is almost dominated by the triplet state of anth, as discussed before. The excitation in the 1MLCT band of 2 also populated the triplet excited state of anth, which, in turn, serves as an energy reservoir extending the relaxation time for the 3 MLCT state,52,54 but also being more external allows easier oxygen bimolecular interaction. This enhancement on 1O2 production was similar to that reported14 for the complex [Ru(dcbpy)2(mbpy-anth)]2+ under the same experimental conditions ΦΔ = 0.76, where it had remarkably increased 1O2 production of [Ru(dcbpy)2(mbpy-COOH)]2+. To evaluate the ability of these complexes to damage DNA, we used a circular DNA, the plasmid pBR322, and monitored the fluorescence response. Because the luminescence signal of the dppz-based ruthenium complexes increased upon intercalation as shown before, we could also assume that after irradiation the bound DNA complexes would generate ROS that might cleave the plasmid in solution. After DNA photocleavage, the dppz ligand becomes more exposed to water molecules, and therefore the luminescence signal should decrease with the irradiation time. We reasoned that, by using this approach, it might be possible to demonstrate a

Figure 8. DPBF consumption (at 20 μmol L−1) as a function of the irradiation time (blue LED) in an air-equilibrated ethanol solution, with and without ruthenium complexes, as indicated (λex = 409 nm).

Figure 9. Photocleavage of 20 μmol L−1 (in base pair) pBR322 DNA in the presence of 1 and 2 at different concentrations in the dark and after 1 h of irradiation with LEDs. For all conditions, lane 1 has a linear DNA ladder and lane 2 has only pBR322 DNA, while lanes 3−6 and 7−10 contained the following concentrations of 0.3, 3.0, 7.0, and 15 μmol L−1 for complexes 1 and 2, respectively. In panel A, samples were kept in the dark, and in panels B−D, samples used blue, green, and yellow LED, respectively. 9091


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Figure 10. Investigation of ROS using scavengers. Photocleavage of 20 μmol L−1 (in bp) pBR322 DNA in the presence of 1 (7 μmol L−1), 2 (7 μmol L−1), and ROS scavengers after 1 h of irradiation with blue LED (463 nm). Lane 1: linear DNA ladder. Lane 2: pBR322 DNA alone. Lane 3: 1 alone. Lanes 4−7: 1 with the following ROS scavengers (at 300 mmol L−1): D-mannitol, NaN3, SOD (20 u), and catalase (20 u). Lane 8: 2 alone. Lanes 9− 12: 2 with the following ROS scavengers (at 300 mmol L−1): D-mannitol, NaN3, SOD, and catalase.

that both complexes cleave DNA mostly by producing 1O2 as a radical product. These data also agree with 1O2 detection using DPBF (Figure 8). Particularly for complex 1, catalase also interfered significantly with photodegradation of the plasmid DNA (Figure 10, lane 7), indicating that peroxide might have a subtle involvement in the photodamage process as well. These results point to a mechanism involving mainly 1O2,56 suggesting mechanisms of types I and II for complex 1 and type II for complex 2. Altogether, these results and the measurement of 1O2 generation support the idea that DNA degradation might be mainly mediated by this radical. Interestingly, a correlation graph of 1O2 generation versus DNA binding showed that our new complexes combined these highly suitable properties (Figure 11), where compound 2 had the highest 1O2 quantum yield along with the strongest DNA binding.

relationship between the ability of the complexes to produce 1 O2 and the luminescence signal of the Ru-DNA system upon irradiation (blue LED, λirr = 463 nm). Accordingly, the complex containing the anthracene moiety presented the largest decrease of luminescence during blue-light irradiation (Figure S13). Therefore, the order of the luminescence decrease was complex 2 > 1 > 3, which corroborated with the 1O2 quantum yield measured. Electrophoresis of DNA Photocleavage Products. To verify any DNA damage as a consequence of binding and ROS generation, suggested before by the fluorescence, we performed agarose gel electrophoresis of the pBR322 DNA under light irradiation (blue LED, λirr = 463 nm; green LED, λirr = 520 nm; yellow LED, λirr = 592 nm). As one control, we used the intercalator 3 under the same conditions (Figure S14). The control lanes without any complex showed undamaged supercoiled pBR322 plasmid (form I; Figure 9A−D, lane 2). There was also no distinct cleavage of pBR322 in the presence of 3 (Figure S11), either in the dark or upon irradiation. Additionally, complexes 1 and 2 incubated in the dark (Figure 9A, lanes 3 and 7) showed no evidence of DNA cleavage. However, these complexes at higher concentrations showed smeared bands from the forms I and II. These smeared bands can result from the formation of intercalated adducts between DNA and complexes, as reported elsewhere.54,55 By contrast, during light irradiation, both complexes promoted a quite distinct effect on the DNA pattern in the agarose gel, compared to 3. These data support the expectation that the pendant ligands employed here indeed had a critical role in the DNA damage. Interestingly, light irradiation with blue and green LED for 1 h showed DNA photocleavage (Figure 9B,C). Unfortunately, irradiation with yellow LED caused only moderate DNA photocleavage. A possible explanation for efficient DNA photocleavage promoted by 1 and 2 could be the extension of DNA−metal complex interaction and/or the higher yield in ROS production. Both complexes 1 and 2 showed enhanced 1 O2 production compared to 3. The production of 1O2 assayed by DPBF (Figure 8) also supported the increased photocleavage activity for 1 and 2. A mechanistic study was carried out to investigate the possible reactive species causing DNA photodamage, where blue-light irradiation was employed. As shown in Figure 10 (lanes 5 and 10), NaN3 had a strong protective effect on the DNA photocleavage caused by complexes 1 and 2, suggesting

Figure 11. Correlation of DNA binding and 1O2 production for complexes 1 (log Kb = 6.83; ΦΔ = 0.66), 2 (log Kb = 6.78 ; ΦΔ = 0.96), 3 (inverted triangle; log Kb = 6.74; ΦΔ = 0.29), 4 (solid circle; log Kb = 3 ; ΦΔ = 0.84), [Ru(dcbpy)2(mbpy-naph)]2+ (open square; log Kb = 3.17 ; ΦΔ = 0.55), and [Ru(dcbpy)2(mbpy-anth)]2+ (open circle; log Kb = 4.7 ; ΦΔ = 0.76), using blue LED.

Antibacterial Activity. Metal-based complexes have been exploited as a novel scaffold for the development of new antimicrobial agents.57−60 One promising antimicrobial strategy, also known as photodynamic antimicrobial therapy, has relied on the photogeneration of ROS, where some ruthenium complexes have been used.61 This process would cause broader damage on many biomolecules (e.g., DNA), leading to 9092


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Inorganic Chemistry microbial death. A series of ruthenium-based complexes have been reported to have enhanced antibacterial activity upon light irradiation, which is associated with ROS generation. We investigated whether our complexes would exhibit any antimicrobial activity either in the dark or upon light irradiation. The MIC and MBC of complexes 1−3 were assayed against bacterial populations of S. aureus, S. epidermidis, P. aeruginosa, and E. coli, as summarized in Table 3. These bacteria were also chosen as a first attempt to sample Grampositive and Gram-negative bacteria.

exhibited antibacterial activity against the Gram-positive bacterium B. subtilis,, but it was inactive against the Gramnegative bacterium E. coli. Bolhuis et al. also reported a series of intercalator ruthenium complexes with activity only on Grampositive bacteria (B. subtilis and S. aureus) but none in Gramnegative (E. coli).62 In contrast to those results, Lei et al. reported great photoactivity for 3 against the Gram-negative bacterium E. coli.66 However, in our hands, neither our complexes nor 3 showed any measurable antimicrobial activity. This could be, in part, due to differences in the sensitivity of the E. coli strains used for those investigations (E. coli JM109), which differed from ours. According to Li et al.,59 the antibacterial activity of their ruthenium complexes can be described as a function of the lipophilicity, charge, and charge separation. The differences in the structures and compositions of the membranes and cell walls of Gram-positive and Gram-negative bacteria could also explain our results. Indeed, Malik et al.67 reported that 1O2 could be effective in killing bacteria, but this action is dependent on a close association with the bacterial membrane. Thus, the outer membrane present in Gram-negative bacteria can constitute an additional barrier that inhibited the antibacterial effect of the ruthenium complexes and prevented their side products from causing cell death. This could be a reason for the lack of activity in Gram-negative bacteria because our antimicrobial activity is also associated with 1O2 generation.

Table 3. Antibacterial Activity of Ruthenium(II) Complexes on Gram-Positive and Gram-Negative Bacteria (Values in μmol L−1) Gram-positive complex

blue LED irradiation

1

ON OFF

2

ON OFF

3

ON OFF

MICa,b MBCc MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

Gram-negative

S. aureus

S. epidermidis

P. aeruginosa

E. coli

6.7 6.7 13.3 13.3 6.4 6.4 6.4 12.8 7.9 7.9 − −

6.7 6.7 6.7 13.3 25.6 25.6 102 102 31.6 31.6 − −

− − − − − − − − − − − −

− − − − − − − − − − − −

■

CONCLUSIONS Our previous approach of coupling a pendant anthracenyl and naphthyl group onto ruthenium(II) bipyridine complexes showed promising enhancement of DNA damage due to better generation of ROS in comparison to an nonmodified complex. A low DNA binding of those complexes, however, limited their selectivity and efficiency as DNA targeting agents. On the basis of these observations, we surmised that a combination of those pendant agents with better DNA binding ruthenium complexes, such as 3, could result in a much more efficient photonuclease agent. Indeed, the incorporation of the organic chromophores naphthalene and anthracene strongly influenced the quantum yield for 1O2 production along with the selectivity for DNA binding. This strategy succeeded in that complexes 1 and 2 can bind very well to DNA, including G-quadruplex DNA, and maintain high 1O2 generation (Figure 9). The combination of good quantum yield for 1O2 production together with selective DNA binding is highly sought out as a route to improving the biological activity, including light-triggered gene knockdown (CALI). DNA degradation was promoted even by yellow LED: an important result that supports the potential use of these complexes as PDT agents. Moreover, complexes 1 and 2 showed promising antibacterial activity on Gram-positive bacteria, following the same order as that of 1O2 generation and DNA binding. The antibacterial activity of these compounds was significantly enhanced with blue LED irradiation, supporting their potential use as PDT agents.

a

Minimum inhibitory concentration. bMIC and MBC are expressed in μg mL−1. (−) Activity not detected even at the highest concentration (complex 1, 0.85 μmol L−1 = 1.00 μg mL−1; complex 2, 0.82 μmol L−1 = 1.00 μg mL−1; [Ru(bpy)2dppz](PF6)2, 1.01 μmol L−1 = 1.00 μg mL−1). cMinimum bactericidal concentration.

In general, the antibacterial effect was stronger when the compounds were irradiated with a blue LED light for 1 h before the plates were incubated (Table 3). Interestingly, complexes 1 and 2 showed reasonably good antimicrobial activity even without light irradiation, supporting the idea that the pendant ligands might bring additional activity. Maybe their better interaction with DNA and single-stranded structures leads to activity. This is particularly interesting compared to the unmodified ruthenium complex, where a complete lack of activity was noticed in the dark. Nonetheless, other ruthenium intercalating complexes showed antimicrobial activity also in the dark.62 In this study, complexes 1−3, when irradiated with blue LED, showed strong DNA binding and generation of 1O2 (Figure 7 and Table 2). Interestingly, our antibacterial results suggested a possible association to 1O2 generation because complex 1 showed lower values of MIC and MBC than 2, followed by 3. Moreover, complexes 1 and 2 induced DNA photocleavage when irradiated with blue LED, but not in the dark (Figures 8 and 9). Altogether, these results support a possible relationship between 1O2 generation, DNA degradation, and antibacterial activity. Beyond this photoactivity, all of the compounds tested were only effective against Gram-positive bacteria. This seems to be a general trend noticed for many other ruthenium(II) complexes used in antimicrobial assays.63−66 Smith et al.64 showed that only upon light irradiation the complex cis-[Ru(bpy)2(INH)2]2+

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b01108. 1 H and COSY 1H NMR, cyclic voltamograms, DFT molecular orbital energy levels and diagrams, UV−vis 9093


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spectra calculated by TD-DFT, emission spectroscopic data, DNA and oligonucleotide structure binding curves, photocleavage of DNA monitored by fluorescence and electrophoresis, and table for selected calculated singlet excited-state transitions for complexes (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Rômulo A. Ando: 0000-0002-3872-8094 Eduardo Henrique Silva Sousa: 0000-0002-0007-8452 Idalina Maria Moreira de Carvalho: 0000-0002-2168-3950 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the CENAPAD-UFC for computer facilities, CENAUREN-UFC for the acquisition of NMR spectra, Adolfo I. B. Romo (UFC) for NMR discussion and interpretation, and the Brazilian agencies CAPES, FUNCAP (Grant PRONEX PR2-0101-0030.01.00/15), and CNPq (Grant 303732/2014-8 to L.G.F.L. and Grant 312030/2015-0 to E.H.S.S.).

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