Chemical and Photochemical Properties of a Ruthenium Nitrosyl Complex with the N-Monosubstituted Cyclam 1-( 3-Propylammonium )-1 , 4 , 8 , 11-tetraazacyclotetradecane

O complexo aminofuncionalizado trans-[Ru(NO)Cl(1-pramcyH)](PF 6 ) 3 (1-pramcyH = 1-(3-propilamônio)-1,4,8,11-tetraazaciclotetradecano) foi sintetizado através da reação do trans[RuCl(tfms)(1-pramcyH)](tfms) 2 (tfms = trifluorometanossulfonato) com óxido nítrico (NO) em solução aquosa ácida. O complexo foi caracterizado por análise elementar, espectroscópica (UVvis, IV, RMN de H e C) e eletroquímica. Dois valores de pK a (7,0 e 8,2) foram determinados para trans-[Ru(NO)Cl(1-pramcyH)](PF 6 ) 3 e foram atribuídos a um dos prótons do grupo amina do cyclam e ao propilamônio. A redução do trans-[Ru(NO)Cl(1-pramcyH)] leva à saída rápida de cloreto seguida de saída lenta de NO, enquanto a irradiação do complexo em solução aquosa desaerada resulta na labilização fotoquímica do NO. O rendimento quântico para a fotoaquação do NO diminui com o aumento do comprimento de onda de irradiação e com a diminuição do pH, e é observável apenas a l irr < 370 nm. O comportamento do trans-[Ru(NO)Cl(1-pramcyH)] é semelhante ao do complexo análogo trans-[Ru(NO)Cl(cyclam)], porém difere daquele do complexo com carboxipropil como substituinte.


Introduction
The discovery of the participation of nitric oxide (NO) in a wide range of physiological processes and pathologies 1 has launched investigations on NO donors, including metal nitrosyl complexes in solution or immobilized in matrices, aiming at the understanding of both fundamental aspects and biological activity for potential applications.  Amo][18]34,[40][41][42][43][44][45][46][47][48][49] These properties can be tuned by the adequate choice of ligands. 14,18 8][39] Because NO can be either beneficial or harmful depending on its bioavailability, compounds capable of releasing NO in a specific biological target have potential biological applications and could be useful tools to study the physiological action of NO.Efforts from this laboratory have been directed toward this goal, using several strategies.One of such approaches involves the functionalization of ruthenium nitrosyl complexes, so they can be linked to important entities such as antibodies and to surfaces, in order to obtain selective NO donor drugs or devices.
In order to verify the effect of the ammoniumpropyl substituent on the configuration of the nitrosyl complex, so as to obtain a wider variety of NO donors, in this paper we report the synthesis, characterization, electrochemical and photochemical properties of the trans-[Ru(NO)Cl(1-pramcyH)](PF 6 ) 3 complex.

Chemicals and reagents
Ruthenium trichloride (RuCl 3 • nH 2 O) (Strem; 40-45% Ru) was the starting material for the syntheses of the ruthenium complexes.Cyclam was purchased from Aldrich.Acetone, chloroform and ethanol were purified according to literature procedures. 52Doubly distilled water was used throughout this work.All other materials were reagent grade and were used without further purification.

Elemental analyses
Analyses were performed at the Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, University of São Paulo, using an Elemental Analyzer CE Instruments, model EA 1110.

Spectra
Electronic absorption spectra were recorded on a Hewlett-Packard model 8452A spectrophotometer using quartz cells.Infrared absorption spectra were obtained in Nujol mulls, in water or in acetonitrile, on a Bomen MB-102 spectrophotometer. 1 H and 13 C NMR spectra were obtained in 5 mm NMR tubes on a Bruker WH400 spectrometer in acetonitrile-d 3 or D 2 O.The EPR experiments were conducted in frozen acetonitrile (77 K) in an ESP-300E Bruker instrument operated with an X-band microwave bridge.
The spectroelectrochemical measurements in the UV-Vis region were carried out in a quartz cell with 0.030 cm optical path, using gold mini-grid, Ag/AgCl and platinum wire as working, reference and auxiliary electrodes respectively.Analogous measurements in the infrared region were carried out using gold mini-grid, Ag wire and gold wire as working, reference and auxiliary electrodes respectively, mounted in a CaF 2 window with 0.020 cm optical path.Successive UV-Vis or IR spectra for trans-[Ru(NO)Cl(cyclam)](PF 6 ) 2 and trans-[Ru(NO) Cl(1-pramcyH)](PF 6 ) 3 were recorded during the reduction process of the complexes at 25 o C, with applied potentials of -500 mV vs. Ag/AgCl in acetonitrile solutions.The pH measurements were performed using a 430 Analion or a Corning pH meters.

Photolyses
Monochromatic irradiations at 313, 334 and 370 nm were carried out using a 150 W Xenon lamp in an Oriel Universal Arc Lamp Source (model 6253).For photolysis at the appropriate wavelengths, the irradiation wavelength was selected with Oriel interference filters, with an average band path of 10 nm.
Considering that the coordinated water of trans-[RuCl(1-pramcyH)(OH 2 )] 3+ has a pK a of 3.1, 48  The calculated f NO values were plotted versus % of the reaction (f x % R).The extrapolated spectroscopic quantum yield at R = 0% was taken as f NO for the photoaquation of NO from trans-[Ru(NO)Cl(1-pramcyH)] 3+ .Evaluation of f NO at R = 0 % eliminates possible complications resulting from secondary photolysis of primary reaction products and inner filter effects.
Because the complex was isolated in acidic medium (0.1 mol L -1 HPF 6 ), the aminopropyl group is protonated.Potentiometric titration allowed estimation of two pK a values (7.0 and 8.2) for trans-[Ru(NO)Cl(1-pramcyH)](PF 6 ) 3 , which should be ascribed to one of the cyclam nitrogen protons and to the protonated propylammonium.Considering that the cyclam nitrogen of the related complexes trans-

and trans-[RuCl(OH)(1-pramcyH)] 2+
have pK a values of 7.9, 8.0, and 7.8, respectively, 48,54 and free propylammonium has a pK a of 9.8, 55,56 it is difficult to undoubtedly assign the pK a values.However, it is more likely that the pK a of 7.0 refers to the cyclam nitrogen and that of 8.2 to propylammonium, value that is lower than 9.8 due to the charge effect of the Ru metal center, as expected.

IR, EPR, NMR and electronic spectra
In nitrosyl complexes, an IR absorption band assigned to NO stretching in the 1950-1800 cm -1 region is associated with a linear structure for Ru-N-O and a nitrosonium character (NO + ). 57Ruthenium complexes of this type are often represented by the resonance form Ru II (NO + ).As pointed out earlier, this formulation is one of several resonance forms (others being Ru III (NO) and Ru IV (NO -)), and, following Enemark and Feltham's 58 notation, the {Ru-NO} 6 complexes are highly delocalized.The IR spectrum of nujol mulls of trans-[Ru(NO)Cl(1-pramcyH)] (PF 6 ) 3 displays three peaks at 1880 cm -1 , 1865 cm -1 and 1842 cm -1 (Figure 1).In the IR spectra of ruthenium nitrosyl complexes recorded from Nujol mulls or KBr pellets, the NO stretching band sometimes appears as two or more peaks or as one peak with one or two shoulders.This feature has been assigned to solid state effects. 18However, only one peak at 1875 cm -1 appears in the spectrum of the aqueous solution of trans-[Ru(NO)Cl(1-pramcyH)] (PF 6 ) 3 ; it is shifted to 1864 cm −1 in acetonitrile (Figure 2), indicating a solvent dependence for the n(NO).These peaks support the nitrosonium character of NO in this complex.An attempt to obtain an EPR spectrum for trans-[Ru(NO) Cl(1-pramcyH)](PF 6 ) 3 showed no signal, giving further support to the IR assignment. ) bound to a cyclam nitrogen.The 1 H NMR chemical shifts in the 5-7.5 ppm range can be assigned to the hydrogen atoms linked to the nitrogens of cyclam and those of the 3-propylammonium group (Figure S1 in Supplementary Information).
Only when the scan range is extended further to -1.5 V (vs.Ag/AgCl) in 0.1 mol L -1 CF 3 SO 3 H/CF 3 SO 3 Na at pH 1, and the coordinated NO 0 undergoes a further reduction process {RuNO} 7/8 (equation 6), or at smaller scan rates, is it possible to observe the presence of the trans-[Ru(H 2 O) 2 (1-pramcyH)] 3+ species (equation 7) in the repetitive scan mode.The trans-[Ru(H 2 O) 2 (1-pramcyH)] 3+ species exhibits a reversible, pH dependent, one electron electrochemical process at E 1/2 = -50 mV close to the ( reported value, at pH 1, of -100 mV 48 and of -155 mV for trans-[Ru(H 2 O) 2 (cyclam)] 2+ . 61The possibility that the Ep 3c process may involve five electrons and result in reduction of NO to amine, as reported for some cases, [62][63][64] is under investigation in our laboratories.
From plots of Ep 2c versus pH it was possible to estimate the equilibrium constants for the reactions represented in equations 8 and 9.

Spectroelectrochemistry
The infrared and UV-Vis spectral changes observed for trans-[Ru(NO)Cl(1-pramcyH)](PF 6 ) 3 when it is submitted to a controlled potential electrolysis at -500 mV vs. Ag/AgCl in acetonitrile (m = 0.1 mol L -1 tba(PF 6 )) are shown in Figures 5 and 6.We were unable to avoid some solvent evaporation during the experiments, which may explain the absence of clean isosbestic points.The intensity of the n(NO) band at 1864 cm −1 decreases, while a new peak appears at 1810 cm -1 , the intensity of which increases.These changes are consistent with the reduction of the nitrosyl ligand, forming trans-[Ru(NO)Cl(1-pramcyH)] 2+ , and are similar to results previously described in the literature. 18,25wever, it has been reported that the spectra of reduced froms of nitrosyl complexes show the n(NO) peak at much lower wavenumbers. 65,66he electronic absorption spectral changes observed for trans-[Ru(NO)Cl(1-pramcyH)](PF 6 ) 3 consist of an absorbance decrease at 263 nm with a concomitant increase at 291 nm.The final electronic absorption spectrum is assigned to the trans-[Ru(NO)Cl(1-pramcyH)] 2+ complex.Likewise, the UV-Vis monitoring of the non-exhaustively controlled potential electrolysis of trans-[RuCl(cyclam) (NO)](PF 6 ) 2 , carried out in the same conditions to promote reduction of NO + to NO, shows absorbance decrease at 254 nm with concomitant increase at 298 nm, which in acetonitrile and under a reductive potential should denote coordinated NO 0 .
The quantum yields pattern for the photolysis of trans-[Ru(NO)Cl(1-pramcyH)](PF 6 ) 3 is similar to those of other nitrosyl ruthenium(am(m)ine) complexes. 14,17,18,46,67he quantum yields decrease as pH decreases and as the irradiation wavelength increases, being noticeable only at l irr < 370 nm.The larger quantum yields achieved at larger pH values can be explained, as in the case of the analogous trans-[Ru(NO)(NH 3 ) 4 (py-X)] 3+ complexes, 46 as follows.The trans-[Ru(NO)Cl(1-pramcyH)](PF 6 ) 3 complex has 3 pK a values, and as the pH increases, in addition to other species a larger fraction of the product is in the hydroxo form.Different species are not necessarily expected to have the same quantum yields.Also, the hydroxo complex is likely to be much less reactive toward the back reaction with NO than the corresponding aquo product, and, thus, has larger quantum yields.As a matter of fact, the increase in quantum yield with larger pH is also consistent with the synthesis of trans-[Ru(NO)Cl(1-pramcyH)] 3+ (see Experimental).   (10)

Conclusions
The procedures described here result in the successful synthesis of a ruthenium nitrosyl complex with the substituted 1-(3-propylammonium)cyclam, trans-[Ru(NO) Cl(1-pramcyH)](PF 6 ) 3 .Unlike 1-carboxypropyl, which results in a fac configuration for the nitrosyl complex, the aminopropyl results in the thermodynamically expected trans arrangement.This amine functionalized complex can be linked to a peptide, or an antibody, for selective biological activity.It can also be bound to a support in a device, such as a stent, to form a drug eluting stent.The behavior of trans-[Ru(NO)Cl(1-pramcyH)] 3+ , which contains a substituted cyclam, parallels that of the cyclam analog, suggesting that these complexes could maintain their properties when linked to a biomolecule.Electrochemical and photochemical experiments suggest the labilization of NO.Like other ruthenium am(m)mine nitrosyl complexes, such as trans-[Ru(NO)(NH 3 ) 4 (py-X)] n+ and trans-[RuCl(NO)(cyclam)](PF 6 ) 2 , trans-[Ru(NO) Cl(1-pramcyH)](PF 6 ) 3 is attractive for potential biological applications because it is stable, water-soluble, and can deliver NO upon activation by reduction at a biologically accessible potential and/or by irradiation with light.Current research in this lab is directed toward these goals for potential applications of the complexes as NO donors.
H NMR spectrum of trans-[Ru(NO)Cl(1-pramcyH)](PF 6 ) 3 in acetonitrile-d 3 shows three signals of NH hydrogens with 3:1:2 intensities (Figure S1).The signal with d 5.36 ppm was assigned to the hydrogen atoms linked to the nitrogen of 3-propylammonium group, while the signals with d 6.06 and d 6.26 ppm were assigned to hydrogen atoms linked to the cyclam nitrogens.These signals were absent in the 1 H NMR in D 2 O because of fast H-D exchange.The integral of the signals assigned to the carbon chain hydrogens of the 1-(3-propylammonium) cyclam ligand is consistent with 26 hydrogens in this molecule (see Experimental section).