Photochemical and Electrochemical Study of the Release of Nitric Oxide from [ Ru ( bpy ) 2 L ( NO ) ] ( PF 6 ) n Complexes ( L = Imidazole , 1-Methylimidazole , Sulfite and Thiourea ) , Toward the Development of Therapeutic Photodynamic Agents

The development of NO photoreleaser compounds has important potential applications on medicine, particularly on preventing topic infections and controlling cancers. Due to these expectations, the photochemical release of nitric oxide from complexes of [Ru(bpy)2LX] , where L = imidazole, 1-methylimidazole, sulphite and thiourea and X = NO and NO2 − was investigated employing spectroscopic and electrochemical techniques. The release of NO was confirmed by chronoamperometry using a NO selective electrode, while the other product, mainly [RuH2O], was detected by UV-Visible spectroscopy and electrochemical techniques for all complexes except for thiourea. The amount of NO released by these complexes upon irradiation was determined using a new developed method using square wave voltammetry.


Introduction
][3][4][5][6][7][8][9][10] The NO molecule is the smallest well-known biological messenger that does not depend on specific transporters. 114][15][16][17][18] Insufficient NO limits NO-mediated signal transduction of normal or protective physiological processes.][21] The molecule of NO reacts with several transition metals producing stable compounds that can be used as source of controlled nitric oxide donors. 22][25][26] The literature describes spectroscopic and photochemical studies of a series of nitrosyl ruthenium complexes, 25,27,28 in particular, the trans-[RuCl( [15]aneN 4 )NO] 2+ species 28 when irradiated with laser of 355 nm in physiologic medium produces NO and the trans-[RuCl( [15]aneN 4 )H 2 O] + species.The quantum yield found for the NO release was high (0.61 mol einstein -1 ) when compared with similar complexes. 27Once nitric oxide is a quite reactive species in biological fluids its therapeutical use requires a controlled and site-specific deliver system to achieve optimum action.2][33] Thus, it is essential an overall investigation of the routes of NO release for its practical use and evaluate if sun light could be used.
A series of ruthenium nitrosyl complexes have been prepared by our group showing promising pharmacological properties such as antiparasitic, 34 brain ischemia/reperfusion protection, 35 inflammatory pain inhibition, 36 and vasodilator. 37At least in some of these studies, there are evidences supporting they may work by increasing NO levels, which activates sGC (soluble guanylate cyclase) pathway signaling, 35 or by inhibiting enzymatic activity through nitrosation reaction. 38Despite these interesting biological data, it is well known that nitric oxide lacks selectivity and shows relative high reactivity in biological fluids.So, there is an increasing interest on developing strategies for selective delivery and release of this key molecule, which could maximize its effect and minimize undesired reactions.Certainly, the use of light is among the most prominent strategy, which has been similarly used in cancer for many decades (photodynamic therapy).As a first step toward the usage of this methodology is to evaluate the photochemical behavior of the therapeutical compounds under controlled photolysis.
In a previous paper 39 we showed that the [Ru(bpy) 2 L(NO)](PF 6 ) n (n = 1 for L = sulfite and n = 3 for L = imidazole, respectively) complexes release NO after NO + /NO reduction mediated by thiols.Also, this complex type showed biological activities such as fungicidal, 34 antiinflammatory, 36 neutroprotector 35 and glyceraldehyde-3phosphate dehydrogenase (GAPDH) inhibitor for Chagas' disease. 38Aiming to evaluate NO release through another mechanistic route, it was carried out photochemical studies of [Ru(bpy) 2 L(NO)] n+ complexes, where L is imidazole (imN), 1-methylimidazole (mimN), sulfite (SO 3 ) and thiourea (tu), and their nitrite species as well.The product of these reactions was followed by UV-Vis spectroscopy and square wave voltammetry technique supporting the analytical use of this later technique for metallonitrosyl investigations in the field.

Chemicals and reagents
Ultrahigh pure water prepared in a Milli-Q system (Direct-Q 3 from Millipore) was employed throughout this work.RuCl 3 .xH 2 O, imidazole, 1-methylimidazole, sulfite and thiourea were obtained from Aldrich and used without further purification.Cis-[Ru(bpy) 2 L(NO)](PF 6 ) n complexes (L = imidazole, 1-methylimidazole, sulfite and thiourea) complexes were prepared according to procedures described in the literature. 23

Apparatus
Samples prepared in buffered solution were irradiated aiming to photorelease nitric oxide, and their quantum yields were calculated based on the amount of photolysed species formed.In these quantitative experiments, it was employed a homemade photochemical reactor UNT 001 containing 355 nm lamps (Figure available in the Supplementary Information), temperature controlled at 25 °C, where the samples were irradiated and the products of these photolysis monitored by square wave voltammetry, following the decrease of the wave at −0.10 to 0.25 V (vs.Ag/AgCl) due to [RuNO] 6/7 redox couple.Qualitative experiments to observe the NO release were also carried out using a NO meter in NO-T model CC (Innovative Instruments).Quantum yield measurements were performed using a Hummingbird diode laser (Quantum Tech) in 377 nm and 419 nm, and product of formation was calculated using a UV-Visible-NIR (near infrared spectroscopy) Hitachi model U-3501 spectrophotometer, using 1.0 cm path length quartz cells until no spectroscopic changes were observed.Electron paramagnetic resonance (EPR) spectra were obtained in a Bruker EMX Plus X-band spectrometer at room or liquid nitrogen temperature.

Electrochemical experiments for determination of NO release
Measurement of the nitric oxide released was accomplished using an electrochemical potentiostat Epsilon (Bioanalytical System Inc.) employing a conventional threeelectrode cell consisting of platinum, glassy carbon, and Ag/AgCl as auxiliary, working and reference electrodes, respectively.These measurements were carried out at 25 °C in a 0.1 mol L -1 aqueous solution of sodium trifluoroacetate (NaTFA), pH 2.0, for L = imidazole, 1-methylimidazole, pH 4.2, for L = thiourea and pH 7.4, for L = sulfite, as supporting electrolyte using square wave voltammetry.The difference in the pH was necessary due to the nitrosyl/nitrite interconversion reaction, which occurs above this pH for each specie (equation 1).According to the equation 1, the pK a are 5.5; 6.4 and 10.3 for L = imidazole, thiourea and sulfite, respectively.
Analytical routine was followed for the development of a quantitative method for measuring the release of nitric oxide.First of all, it was established the parameters that showed the best results for current vs. concentration relationship by varying frequency (10-90 Hz), width (10-70 mV) and increment (1-10 mV) of the wave, where only one parameter was changed each time during optimization (analytical curves are available in the Supplementary Information).
Analytical standard curve was prepared using nitric oxide metal complexes by taking the current value at the peak for the NO +/0 redox process for each concentration, which resulted in a linear relationship.
A solution of each nitrosyl compound was irradiated and the amount of NO released was monitored with successive electrochemical measurements of the current at the peak of the potential of the coordinated NO +/0 redox couple.Once, these voltammograms were analyzed and peak currents extracted at each time point, the NO concentration were calculated by interpolation of the standard curve, prepared previously.Since the stoichiometric relationship between NO released and bound to the complex is the same, then the difference in NO concentration before and after the irradiation is equal to the concentration of NO released in the process.

Results and Discussion
The absorption spectra of the cis-[Ru(bpy) 2 L(NO)] n+ nitrosyl complexes, where L = imidazole, 1-methylimidazole, sulfite and thiourea, showed bands from 310-326 nm attributed to (bpy)π*←dπ(Ru) MLCT transition.The (NO + )π*←dπ(Ru) transitions were not detected in the spectra probably because it is overlapped by the (bpy)π*←dπ(Ru) transitions.][42] The wavelength used in the irradiation of the complexes was chosen according to their absorption bands.In aqueous solution ruthenium nitrosyl complexes exhibit the equilibrium displayed in the equation 1: 23,43,44 [Ru(bpy where for the complexes investigated here the pK a values for this equilibrium are 10.3, 5.5, and 6.4 for L = SO 3 2− , imidazole and thiourea, respectively. 23So, for these studies it was chosen a pH where only one species is predominant, such as in the RuNO + form.
Photochemical release of nitric oxide has been widely investigated during the last years as a selective strategy to deliver this pharmacological relevant molecule; however; still several drawbacks prevent its suitable use.There are several studies concerning photochemical reactivity of ruthenium nitrosyl complexes when irradiated in the ultraviolet and visible range.Interestingly, many of these reports has suggested that NO release is a consequence of the photoelectronic energy transfer from the metal center to the nitrosyl species forming the oxidized metal center and reduced NO as final products as illustrated in equation 2. 25,26,[45][46][47][48] However, Ru III -H 2 O species is not always observed as a photoproduct, where in some cases it has been detected Ru II -H 2 O species instead.Rose and Mascharak 45 had proposed that the photochemical behavior of the {RuNO} 6 regarding to the NO release could be classified in two types of mechanisms: the first one is observed the formation of the NO and [Ru III -solvent], while the second one is observed the formation of NO and [Ru II -solvent] as final products.The first type is noticed for the Ru-nitrosyl complexes having as auxiliary ligands ammine, carboxamide, thiolate, heme and phenolate, whereas the second type for polypyridines and Schiff base as auxiliary ligands.For the second type of mechanism, despite it is not very clear, the formation of Ru II species seems to proceed from a spontaneous reduction of the transient Ru III species caused by incident light. 45,46In our case, the irradiations conducted at 377 nm (for nitrosyl complexes) and at 477 nm (for nitrite complexes) for the solutions of the complexes with L = SO 3 2− , imidazole and 1-methylimidazole showed profiles compatible to Ru II -H 2 O species as supported by UV-Vis, EPR and 1 H NMR (nuclear magnetic resonance) spectra (data available in the Supplementary Information).These results suggested that these complexes follow the second type of mechanism proposed by Rose and Mascharak. 45To confirm this, these complexes were fully reduced by Zn/Hg amalgam and the spectrum recorded (Figure 1) was the same obtained after photolysis, as described above.Also, the 1 H NMR spectrum of the product of irradiation at 350 nm for the sulfite complex, showed a profile compatible with the [Ru(bpy) 2 (SO 3 )(H 2 O)] 2+ complex ion.Additionally, the EPR spectrum was collected for a solution containing the [Ru(bpy) 2 (SO 3 )NO] + after 60 minutes of laser irradiation at 350 nm and it did not show any significant signal of paramagnetic Ru III , which is compatible with a EPR silent species such as Ru II -H 2 O as proposed here (the spectrum is available in the Supplementary Information).The ruthenium complexes containing L = imidazole and 1-methylimidazole showed similar behavior, where their photoproducts were also EPR-silent consistent with Ru II -H 2 O.However, for the complex containing thiourea as auxiliary ligand, the electronic spectrum profile suggested the formation of another species instead the Ru II -H 2 O, which was not fully characterized and is still under investigation.Nevertheless, this photoproduct was also EPR-silent supporting Ru II is maintained as final species.

F i g u r e 2 s h ow s s p e c t r a l c h a n g e s f o r t h e
cis-[Ru(bpy) 2 (imN)(X)] n+ complexes, where X = NO 2 − and NO + , before and after irradiation for 30 min at 447 nm and 377 nm, respectively.After irradiation, both solutions exhibited a decreasing in the absorbance of the bands at 433 nm for NO 2 − followed by the appearance of a new band at 488 nm for both complexes in an agreement with the formation of the same specie.
Silva and co-workers 46,47 and Rose and Mascharak 45 reported photochemical studies of a similar ruthenium nitrosyl complexes.They proposed upon irradiation an electron transfer from the metal center to the nitrosyl ligand occurs generating an intermediate species with a consequent NO release and formation of the [Ru III -OH 2 ] which is further reduced forming as final product [Ru II -OH 2 ] moiety, besides the nature of the reducing agent being unknown, with the suggestion that the NO also can act in this process.This observation is the same as detected in the compounds presented here (equations 3-5).(5) Since free NO was verified by the NO meter (Figure 3) and the final product obtained from the irradiation showed similar electronic spectra to [Ru II -OH 2 ] species, a mechanism of monoelectronic reduction is likely for all the nitrosyl species, except the [Ru(bpy) 2 (tu)NO] 3+ , which   is observed the formation of a different final species, as mentioned before, not identified yet.
Quantitative data is presented in Table 1 which summarizes the quantum yield for the formation of the [Ru II -OH 2 ] species after irradiation of the nitrosyl complexes type [Ru(bpy) 2 LNO] n+ presented in this work.This measurement assumes that the formation of the [Ru II -OH 2 ] and NO release are equimolar.It is important to remark that for all of the complexes, quantum yield measurement was based only on the formation of the aquo species as detected by electronic absorption, and free NO was detected qualitatively by NO meter (Figure 3).
As the pH for these experiments was kept in 2.0-7.4 range, for the [Ru(bpy) 2 (SO 3 )(NO 2 )] − it was not possible to proceed this since in this pH range this compound is presented in nitrosyl form instead of nitrite form. 23he data collected in this work can be compared to similar compounds prepared elsewhere, cis-[Ru(bpy) 2 (L)(NO 2 )] + , where L = pyridine, 46 4-pycoline 46 and pyrazine 46 (Table 1).Despite the usage of a much longer wavelength, the results presented here were still considerably attractive granting further attempt to modulate compounds which can act as NO donors under irradiation. 46Here, it was observed the formation of the aqua complex for all the species upon irradiation in agreement to their electrochemical and spectroscopic profiles.However, the best nitric oxide photo releaser compound was found to be the complex [Ru(bpy) 2 (1-meimN)NO](PF 6 ) 3 which was directly followed by the [Ru(bpy) 2 (1-meimN)NO 2 ]PF 6 .The other compounds also present significant photochemical release of nitric oxide in the same way.
In order to quantify the amount of NO released, for the nitrosyl complexes, the photochemical release of NO was also monitored by square wave voltammetry following the decrease of the peak current centered at −0.10 to 0.25 mV range attributed to the coordinated NO +/0 redox process (Figure 4).By observing the linear correlation between the peak current and the complex concentration (R = 0.999, Figures available in the Supplementary Information) it was possible to calculate the amount of NO released after irradiation (see Experimental section).Table 2 summarizes the results of the NO release after 10 min of irradiation, where it can be observed the co-spectator ligand 1-methylimidazole (1-meimN) promotes a better NO releasing if compared to the worse process for sulfite complex, being the trend of the percentage on NO released the same as the quantum yield.
These results are in good agreement with the quantum yield data, where the highest φ for the formation of the Ru II -OH 2 species was observed for the [Ru(bpy) 2 (1-meimN)NO] 3+ complex, which also presents the highest percentage of NO release quantified by square wave voltammetry (87.1%).Interestingly, this new approach to quantify indirectly

Table 2 .
Results of the calculation of the liberation photochemical of nitric oxide for the cis-[Ru(bpy) 2 L(NO)] n+ complexes, using the technique of square-