Interactions between DNA Purines and Ruthenium Ammine

J. Phys. Chem. B 2010, 114, 3987–3998

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Interactions between DNA Purines and Ruthenium Ammine Complexes within Nanostructured Sol-Gel Silica Matrixes Luı´s M. F. Lopes,† Ana R. Garcia,†,‡ Pedro Brogueira,§ and Laura M. Ilharco*,† CQFM - Centro de Quı´mica-Fı´sica Molecular and IN - Institute of Nanoscience and Nanotechnology, Instituto Superior Tećnico, UniVersidade Tećnica de Lisboa, AV. RoVisco Pais 1, 1049-001 Lisboa, Portugal, Departamento de Quı´mica e Farmaćia, FCT, UniVersidade do AlgarVe, Campus de Gambelas, 8005-139 Faro, Portugal, and Departamento de Fı´sica and ICEMS, Instituto Superior Tećnico, UniVersidade Tećnica de Lisboa, AV. RoVisco Pais 1, 1049-001 Lisboa, Portugal ReceiVed: August 20, 2009; ReVised Manuscript ReceiVed: February 8, 2010

The interactions between DNA purines (guanine and adenine) and three ruthenium ammine complexes (hexaammineruthenium(III) chloride, hexaammineruthenium(II) chloride, and ruthenium-red) were studied in a confined environment, within sol-gel silica matrixes. Two encapsulation methods were rehearsed (differing in temperature and condensation pH), in order to analyze the effects of the sol-gel processes on the purines and on the Ru complexes separately. The extent of decomposition of the Ru complexes, as well as the interactions established with the purine bases, proved to be determined by the coencapsulation method. Combined results by diffuse reflectance UV-vis and infrared spectroscopies showed that, when coencapsulation is carried out at 60 °C, specific H bonding interactions are established between the amine group of Ade and the ammine groups of the Ru complex or the hydroxo group of an early decomposition product. These are responsible for the important role of the purine in inhibiting the oxidation reactions of the Ru(II) and Ru(III) complexes. In contrast, Gua establishes preferential H bonds with the matrix (mainly due to the carbonyl group), leading to higher yields in the final oxidation products of the Ru complexes, namely, trimers and dimers. Direct covalent bonding of either purine to the metal was not observed. 1. Introduction Ruthenium coordination complexes have been receiving great attention due to their new and unique catalytic characteristics1 and to their promising performance in the medical field,2 in particular as an alternative to platinum tumor-inhibiting agents. A few compounds are presently standing clinical trials (phase II), namely, NAMI-A (imidazolium [trans-tetrachloro(1Himidazole)(S-dimethylsulfoxide)ruthenate(III)])3 and KP1019 (indazolium [trans-tetrachlorobis(1H-indazole)ruthenate(III)]).4,5 Although new cancer treatment strategies have been evolving toward specific pathways, notably those involved in cell signaling,6 the majority of cytotoxic metal complexes available and under research have as a target the DNA, given its importance in replication and cell viability. In fact, the mutagenicity and anticancer activity of coordination complexes have stimulated the study of the interactions of a wide range of compounds with nucleic acids,7 nucleotides,8 nucleosides,9 their constituent bases and derivatives,10 as well as other biological targets such as proteins,11 in an attempt to elucidate their biochemical mechanisms and understand the role of the metal center in nucleic acid metabolism. However, most of these studies were conducted in solution and so the influence that a restricted environment has over the structure and reactivity of these molecules has been overlooked. * Corresponding author. Phone: +351-218419220. Fax: +351-218464455. E-mail: [email protected]. † CQFM - Centro de Quı´mica-Fı´sica Molecular and IN - Institute of Nanoscience and Nanotechnology, Instituto Superior Tećnico, Universidade Tećnica de Lisboa. ‡ Universidade do Algarve. § Departamento de Fı´sica and ICEMS, Instituto Superior Tećnico, Universidade Tećnica de Lisboa.

Figure 1. Structures of (A) hexaammine Ru(II) and hexaammine Ru(III) cations and (B) ruthenium-red.

The coordination of transition-metal compounds offers many binding modes to polynucleotides, including outer-sphere noncovalent binding, metal coordination to nucleobase and phosphate backbone sites, as well as strand cleavage induced by oxidation using redox-active metal centers.12 It has been shown that ammine complexes of Ru(II) and Ru(III) and their related aquo products tend to selectively bind to imine sites in biomolecules, mainly through coordination to histidyl imidazole nitrogens on proteins and the N7 site on the imidazole ring of purine nucleotides and nucleic acids.2 Among purine bases, preferential affinity for the guanine N7 site has been detected,13 although metal migration to other nucleobases may occur.14 The physical and chemical effects of the metal ion strongly coordinated to different sites of the purine ring have also been reported.15 When cationic complexes are involved, it would be expected that interactions with DNA occur through the negatively charged phosphate backbone. In general, Ru(II) and Ru(III) hexaammine complexes (Figure 1A) have octahedral geometry stabilized by the strong ligand field.16 The assumption of an Oh symmetry is reasonable for solution, but the crystal-site symmetry in the solid state may

10.1021/jp9080542  2010 American Chemical Society Published on Web 03/02/2010

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be lower.17 Both [Ru(NH3)6]2+ and [Ru(NH3)6]3+ are efficient electron donors.18 Ru(II) and Ru(III) centers show contrasting characters with regard to π-symmetry interactions with coordinated ligands; i.e., 4d5 Ru(III) is a good π acceptor,19 while 4d6 Ru(II) is a good π donor. Thus, the ligands in its coordination sphere are quite labile.20 In fact, the powerful reductor hexaammineruthenium(II) complex is very unstable even in the presence of air and moisture, decomposing to Ru(III) hexaammine and Ru0. In aqueous solution, this disproportionation reaction is accompanied by the release of ammonia:21 3[Ru(NH3)6]2+ f 2[Ru(NH3)6]3+ + Ru0 + 6NH3

(1)

In basic environment, the low spin d5 hexaammineruthenium(III) complex undergoes a very fast proton exchange reaction, presumably as a consequence of the high acidity of these ions:22 [Ru(NH3)6]3+ + OH- f [Ru(NH3)5NH2]2+ + H2O

(2)

The deprotonation increases the instability of the ligands and the susceptibility to undergo an aquation reaction, which eventually leads to the formation of the ruthenium trimer [RuIII(NH3)5ORuIV(NH3)4ORuIII(NH3)5]6+ (ruthenium-red): [Ru(NH3)5NH2]2+ + H2O f [Ru(NH3)5OH]2+ + NH3

(3)

[Ru(NH3)5OH]2+ + H2O f trans-[RuIV(NH3)4(OH)2]2+ + 1/2H2 + NH3 (4) 2[Ru(NH3)5OH]2+ + trans-[RuIV(NH3)4(OH)2]2+ f [Ru(NH3)5ORu(NH3)4ORu(NH3)5]6+ + 2H2O (5) Other mechanisms have been suggested for the decomposition of this Ru complex.23 These reactions have been the object of extensive studies in solution,21,23–25 in zeolites,26–28 and in other micro- and mesoporous supports.29–31 It was shown that the stability of the [Ru(NH3)6]3+ complex can be drastically affected by the pore structure (dimensions and geometry), framework basicity, and atmosphere nature, as well as by the level of hydration.32 Such structural and textural characteristics of the support are particularly relevant, since the reactions involved in the decomposition of [Ru(NH3)6]3+, namely, toward the formation of ruthenium trimers, are dependent on the available space and mobility of the molecules inside the porous network, and on the presence of a favorable chemical environment. It was proved that below 100 °C Ru-red is formed in vacuum, in air, and in water vapor, but it is largely suppressed under O2 and NO atmospheres.26 Any molecule which favorably competes with H2O to enter the coordination sphere or to react with coordinated NH3 suppresses the formation of Ru-red. Above 100 °C, a new decomposition product was detected, a nitrosyl Ru complex, whose concentration is environment dependent in the following order NO > O2 > H2O > vacuum.26 Ruthenium-red (Figure 1B) has two linear oxo-bridges to satisfy the hexa-coordination of ruthenium atoms. When the NH3 ligands are in an eclipsed configuration, the proposed symmetry is D4h.33 The antitumoral properties of Ru-red were discovered in the 1970s, based on the growth inhibition of Lewis lung carcinoma

in mice, due to the inhibition of Ca2+ transport both at the mitochondrial and at the cell membrane level.34,35 Its binding to a wide range of anions, including RNA, DNA, phospholipids, and acidic peptides, was demonstrated.36,37 Extensively studied by its remarkable immunosuppressant activity,38 it has also been used as a dye in histology and electron microscopy, but rarely for catalytic purposes as a metal precursor of ruthenium catalysts.39 Similarly to the other ruthenium ammine complexes, Ru-red has a limited stability and undergoes hydrolysis in acidic solution and when exposed to air and moisture, resulting in a mixture of Ru-red and its one-electron oxidation product, Rubrown, [RuIV(NH3)5ORuIII(NH3)4ORuIV(NH3)5]7+.40 Ru-red may also undergo thermal and photodecomposition, whereas Rubrown exhibits a high thermal and photochemical stability, possibly due to the charge delocalization among the ruthenium centers through the oxo-bridges (RuIV-O-RuIII-O-RuIV).41 Other common decomposition products from Ru-red are the µ-oxo ruthenium dimers of the form [(NH3)5RuORu(NH3)5]5+/4+,42 or [X(NH3)4RuIIIORuIV(NH3)4X]3+, where X ) OH- or Cl-, known as Ru360,2 which often appears as an impurity in commercial preparations. Silica-based materials, in particular xerogels, have been used for a long time as support matrixes for drugs, due to some important characteristics: they are nontoxic and biocompatible, causing no adverse tissue reactions, and degrade in the body to silicic acid (Si(OH)4), which is eliminated through the kidneys.43–45 The simplicity, versatility, and mild characteristics of the sol-gel process turn it into a useful method to prepare silica materials of high purity through the hydrolysis and condensation of suitable metal alkoxides, allowing the introduction of metal complexes within the inorganic network.46 It is the aim of the present work to analyze the interactions between ruthenium ammine complexes and DNA purines, in a confined environment created by nanostructured sol-gel silica matrixes. For that purpose, [Ru(NH3)6]Cl2, [Ru(NH3)6]Cl3, Rured, guanine, and adenine were separately encapsulated and then coencapsulated in the initial sol mixture, and their molecular structures within the xerogel analyzed by UV-vis and infrared spectroscopies. 2. Experimental Section 2.1. Materials. Hexaammineruthenium(III) chloride ([Ru(NH3)6]Cl3, Ru 32.1% min), hexaammineruthenium(II) chloride ([Ru(NH3)6]Cl2, 99.9%), and ruthenium(III) chloride oxide ammoniated or tetradeca-ammine-di-µ-oxo-triruthenium hexachloride ([Ru(NH3)5ORu(NH3)4ORu(NH3)5]Cl6, or Ru-red, Ru 35.3% min) were supplied by Alfa Aesar. Tetraethoxysilane (TEOS, 98%), adenine (99%), and guanine (99.9%, pure) were purchased from Sigma Aldrich. The catalysts used in the sol-gel process were hydrochloric acid (HCl) from Riedel-de-Hae¨n and ammonium hydroxide (NH4OH) from Merck. 2-Propanol (99.8%) was obtained from Fluka. All chemicals were used without further purification. Distilled water was used in the sol-gel synthesis and in the preparation of aqueous solutions. 2.2. Preparation of the Sol-Gel Monoliths. The synthesis consisted of a two-step sol-gel process essentially reported elsewhere for the control silica.47,48 Two encapsulation procedures were followed, in which the Ru complex was incorporated in the sol-gel mixture before prehydrolysis, at 60 °C (method I), and after prehydrolysis, at 20 °C (method II). 2.2.1. Method I: Encapsulation of the Ru Complex before Prehydrolysis, at 60 °C. The appropriate amount of ruthenium complex was weighed to a polypropylene container and dissolved with 0.08 M HCl (6.50 mL), under stirring, until a

DNA Purines and Ruthenium Ammine Complexes

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TABLE 1: Samples Prepared by Methods I and IIsNature of the Dopant(s), Relevant Molar Ratios, and Gelation Times (tG) method I

method II

molar ratios purine/ TEOS

sample control SiO2 Ade/SiO2 Gua/SiO2 [RuII(NH3)6]Cl2/SiO2 [RuII(NH3)6]Cl2/Ade/SiO2 [RuII(NH3)6]Cl2/Gua/SiO2 [RuIII(NH3)6]Cl3/SiO2 [RuIII(NH3)6]Cl3/Ade/SiO2 [RuIII(NH3)6]Cl3/Gua/SiO2 [RuIII(NH3)5ORuIV(NH3)4ORuIII(NH3)5]Cl6/SiO2 [RuIII(NH3)5ORuIV(NH3)4ORuIII(NH3)5]Cl6/Ade/SiO2 [RuIII(NH3)5ORuIV(NH3)4ORuIII(NH3)5]Cl6/Gua/SiO2

complex/ TEOS

molar ratios purine/ complex

tG (min) 7 15 17 3 2 2 10 16 22 25 26 28

0.016 0.012 0.016 0.012 0.016 0.012 0.015 0.012

0.0096 0.0096 0.0099 0.0094 0.0095 0.0095 0.0004 0.0003 0.0002

1.71 1.22 1.72 1.27 49.67 61.50

purine/ TEOS

complex/ TEOS

purine/ complex

0.021 0.011 0.021 0.011 0.021 0.011 0.021 0.012

0.0098 0.0099 0.0099 0.0091 0.0091 0.0091 0.0005 0.0004 0.0004

2.12 1.13 2.31 1.23 52.50 30.00

TABLE 2: Observed Color Changes during Sample Preparation by Method I color Ru complex

powder

initial solution

during hydrolysis

upon neutralization

after aging

after drying

[RuII(NH3)6]Cl2 [RuIII(NH3)6]Cl3 [RuIII(NH3)5ORuIV(NH3)4ORuIII(NH3)5]Cl6

black yellow purple

pale yellow colorless pale purple

yellow colorless brown-red

darker yellow pale yellow pale purple

purple pale purple red-brown

dark brown dark brown brown

homogeneous solution was obtained. Then, 2-propanol (6.30 mL), the cosolvent, was added and the mixture vigorously stirred. Finally, the silica precursor, TEOS (2.00 mL), was added dropwise, under stirring. The container with the reaction mixture was sealed and placed in an oven at 60 °C, under stirring (140 rpm), for 60 min. After that period, the mixture was gently neutralized by addition of NH4OH (2.6 mL) and left to gel without stirring, at the same temperature. The time elapsed between the neutralization and gelation of the mixture (gelation time, tG) was recorded. To strengthen the silica network, the gels were left to age for 48 h at 60 °C, in the residual liquid. This was then removed and the samples left to dry: 1 week with the container partially covered at 60 °C and 2 weeks open (the first one at the same temperature and the second at 75 °C). The monoliths obtained (small cylinders of ∼2 cm in diameter and ∼1 cm in height) were characterized and stored at ∼35 °C, under a partially dried atmosphere. In the preparation of monoliths impregnated with adenine, a similar procedure to the one just described was followed, but the Ru complex was replaced by the appropriate amount of adenine. Given the lower solubility of guanine, a 0.017 M stock solution of this purine was prepared in 0.08 M HCl at ∼90 °C, under vigorous stirring, until a colorless and translucent solution was obtained (∼1 h). It was left to cool to room temperature and then used in the sol-gel synthesis instead of the 0.08 M HCl solution. To synthesize the monoliths with coencapsulated adenine and Ru complex, the required amounts were weighed and dissolved in 0.08 M HCl, and this solution used as described above. For coencapsulating guanine and Ru complex, the stock solution of guanine was used as the solvent for the powder Ru complex. In all of the samples, the H2O/TEOS, 2-PrOH/TEOS, HCl/ TEOS, and NH4OH/HCl molar ratios were kept constant at 40, 9.2, 0.058, and 1.00, respectively. The other relevant molar ratios are listed in Table 1. The very low solubility of Ru-red imposed the reduced concentrations of this Ru complex. Significant color changes were observed for the samples prepared by method I throughout the sol-gel stages, as summarized in Table 2.

These changes suggest that the Ru(II) and Ru(III) complexes undergo oxidation reactions toward Ru-red and Ru-brown during the sol-gel process, with a certain delay for the Ru(III) complex. Encapsulation method II was followed in an attempt to stabilize these Ru complexes. 2.2.2. Method II: Encapsulation of the Ru Complex after Prehydrolysis, at 20 °C. The temperature was kept at 20 °C throughout the whole process from encapsulation of the Ru complexes to drying. The prehydrolysis was carried out following the procedure described in section 2.2.1. The mixture was then cooled to 20 °C, under continuous stirring, and gently neutralized by addition of NH4OH (2.6 mL). The required amount of Ru complex was directly dissolved, and the mixture was immersed in an ultrasound bath for 30 s and then left to gel without stirring. The aging period was 48 h, and drying took 3 weeks: the first one at ambient pressure and the last two under vacuum (10-3 mbar). For encapsulation and coencapsulation of the purines, a stock solution of each base was used as a substitute for the 0.08 M HCl solution: 0.016 M in guanine, prepared as described in 2.2.1, and 0.030 M in adenine, prepared at room temperature in an ultrasound bath. In all of the samples, the H2O/TEOS, 2-PrOH/TEOS, HCl/TEOS, and NH4OH/HCl molar ratios were kept constant at 40, 9.2, 0.058, and 1.30, respectively. The molar ratios regarding the purine bases and Ru complex contents are indicated in Table 1. 2.3. Characterization. The structural analysis by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy was performed using a Mattson RS1 FTIR spectrometer with a Specac Selector, in the range 4000-400 cm-1 (wide band MCT detector), at 4 cm-1 resolution. The spectra are the result of 500 added scans for each sample, ratioed against the same number of scans for the background (ground KBr, FTIR grade from Aldrich). All solids (monoliths and powders) were previously grinded and mixed with potassium bromide in appropriate proportions to obtain spectral absorbance in the range of applicability of the Kubelka-Munk transformation.49 The UV-visible spectra were recorded in transmission mode (for solutions) using a Jasco model V-650 spectrophotometer

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Figure 2. (A) Atomic force microscopy image of the sample [Ru(NH3)6]Cl2/SiO2 prepared by method I and (B) optical microscopy image of the same sample; (C) optical microscopy image of the control silica.

and in diffuse reflectance mode (for powder samples) in a Shimadzu UV-3101PC double beam spectrophotometer coupled to a MPC-3100 unit equipped with an integrating sphere coated with BaSO4. The baseline was recorded for BaSO4 (Wako Pure Chemical Industries). The spectral range between 200 and 900 nm was scanned. Ground state absorption spectra in terms of the remission function were obtained using the Kubelka-Munk equation. A Dimension 3100 microscope with a Nanoscope IIIa controller from Digital Instruments was used for atomic force microscopy (AFM) measurements. These were performed in tapping mode under ambient conditions. A commercial tapping mode etched silicon probe from DI and a 90 µm × 90 µm scanner were used. Optical microscopy images were captured from a Sony CCD XC-999P video camera C-mount coupled to a single tube microscope with a 10× Nikon objective. The scale was calibrated by imaging a standard sample with 10 µm × 10 µm squares. 3. Results and Discussion 3.1. Gelation Times and Microscopic Observation. From the data in Table 1, it becomes clear that encapsulation of a purine base, [Ru(NH3)6]Cl3, Ru-red, or purine/Ru complex pairs, by method I, has an inhibiting effect on the sol-gel reactions that results in longer gelation times (tG) than those for the control silica. Ru-red, although in a lower concentration than all of the other dopants, induces the largest delay (from 7 to 25 min). This may be due to a spatial hindrance to the condensation of the silica oligomers. On the other hand, two opposing electrostatic effects may occur: the positive ions may screen the charges of the negative silica particles (the pH of the medium is above the isoelectric point of silica), but a large number of Cl- ions per Ru complex molecule is also present in the medium. The two purines have similar effects, although the samples with Gua exhibit slightly higher tG than those with Ade. The Ru(II) complex is the exception, as it induces a much shorter gelation time, suggesting a pronounced catalysis of the condensation reaction. This particular behavior may derive from the higher lability of the ammine ligands due to the d6 configuration of the metal, enhanced by temperature (60 °C): this Ru complex may release NH3 (eq 1), raising the pH of the medium and consequently catalyzing condensation. In fact, despite the constant NH4OH/HCl molar ratio, the pH of the samples with [Ru(NH3)6]Cl2 resulted in the range 7-8, instead of 6-7 obtained for all of the other Ru complexes. Additionally,

the color change pointed to the presence of Ru(III) species. The combination of AFM and optical microscopy images of the sample [Ru(NH3)6]Cl2/SiO2 shows the coexistence of two phases: an amorphous material (Figure 2A) and crystalline agglomerates of 10-20 µm, beyond the scope of AFM (Figure 2B). These observations confirm that the disproportionation reaction (1) takes place during the sol-gel process, leaving oxidized [Ru(NH3)6]3+ species within the silica matrix and Ru0 as bright metallic clusters. It is interesting to note that, although these Ru cluster dimensions would be too large for catalytic purposes, they could be optimized just by controlling the sol-gel conditions, without need for the characteristic reduction conditions used in the preparation of supported catalysts.50 The catalytic effect of this Ru complex on condensation is so strong that even when it is coencapsulated with the purine bases the gelation time stays extremely short. For the samples synthesized by method II, the nature of the doping molecule does not determine the gelation times, since all of the samples took 7-10 min to gel. This may be explained by the higher NH4OH/HCl molar ratio used (1.30) that prevails over any other perturbation. 3.2. Effects of Encapsulation on the Ru Complexes. The effects of encapsulation on the structure of the Ru complexes were studied by comparing the absorption spectra in solution with the diffuse reflectance spectra in the solid phase, in the UV-vis, and infrared regions. The UV-vis spectra are shown in Figure 3 and the results summarized in Table 3. Assuming an eclipsed D4h structure for the Ru-red cation, the ground-state configuration (egb)4(eub)4(eg)4(eu*)4(b2gb)2(b1u)2(b2g*)2(eg*)0 has been proposed.33 However, the b molecular orbitals are virtually degenerate and their energies relative to eg and eu* orbitals are uncertain, having led to the alternative proposal (egb)4(eub)4(b2gb)2(b1u)2(b2g*)2(eg)4(eu*)4(eg*)0.51 As a consequence, there is some controversy in the literature concerning the spectral assignment.33,51,52 The strong band at 534 nm observed in basic aqueous solution (Figure 3, lower curve, gray) is unambiguously attributed to the z polarized eg* r eu* transition. The remaining features were assigned in agreement with the first ground state configuration, as indicated in Table 3. The band at 370 nm emerges due to perturbations of the local octahedral geometry of the metal atom in ruthenium trimers;31 an alternative assignment has been proposed for a monomeric water-substituted Ru complex formed by the photodecomposition of Ru-red.41 In acidic solution, the strong high energy band shifts to 255 nm, the one at 370 nm deforms toward lower wavelengths, a new component appears at



Figure 3. UV-vis diffuse reflectance spectra (upper) of Ru complexes encapsulated in a silica matrix by methods I (gray) and II (black), normalized to the maximum and subtracted for the very weak spectrum of the control silica. UV-vis absorption spectra (lower) of Ru complexes in fresh aqueous solution, normalized to the maximum: basic (gray) and acid (black).

TABLE 3: Assignment of the UV-vis Spectra of the Ru Complexes in Solution and Encapsulated within Silica Matrixes sample Ru-red

[Ru(NH3)6]Cl3

[Ru(NH3)6]Cl2

aq. sol. (basic) aq. sol. (acid) /SiO2 (method I) /SiO2 (method II) aq. sol. (acid) aq. sol. (basic) /SiO2 (method I) /SiO2 (method II) aq. sol. (acid) aq. sol. (basic) /SiO2 (method I) /SiO2 (method II)

assignments25,30,33 a

wavelength (nm) 261vs 255vsa 268vs 261vs

342sha 276vs 276vs ∼273sh 295vsb 263vs 263vs

eg* r eub

∼330sh

370w 361w 368m 368w

482ma 482wa 484ma

534s 532m

741w 741w

536s

762m

400w

266vs

339vs 330vw ∼347sh ∼347sh 347vsa 333m

LMCT

d-d

463wa

481sa ∼480vw

367vs eg*reg

LMCTc

eg*reu*

eg*reu*

eg*rb1u

Ru-brown. b In the intermediate [Ru(NH3)5OH]2+. c In the intermediate [Ru(NH3)5NH2]2+.25,30

481 nm, and the relative intensity of the band at ∼534 nm decreases. This pattern is consistent with a partial conversion of Ru-red to Ru-brown ([RuIV(NH3)4ORuIII(NH3)5ORuIV(NH3)4]7+).41 In fact, the absorption spectrum of Ru-brown in acid aqueous medium (not shown) is quite similar to that of Ru-red but shifted to higher energies (255, 350, and 465 nm), with the exception of the eg* r b1u transition that shifts to ∼885 nm.33 The spectrum of encapsulated Ru-red (Figure 3, upper) strongly depends on the encapsulation method: by method II, the component at 484 nm suggests some oxidation of Ru-red to Ru-brown, whereas, by method I, the characteristic Ru-red bands at 536 and 762 nm are suppressed, with only the Rubrown fingerprint remaining at 482 nm. Besides, a new component appears as a shoulder at 342 nm, whose nature is discussed below. This oxidation to Ru-brown during the sol-gel process is consistent with the observed color change toward brown (Table 2). The UV-vis absorption spectrum of [Ru(NH3)6]Cl3 in aqueous acid solution (Figure 3, lower, black) is similar to the diffuse reflectance spectrum obtained for the complex encapsulated at 20 °C (by method II): they are both dominated by a strong NH3 f Ru(III) charge transfer (LMCT) band,25 with a maximum at 276 nm in solution, which broadens and shifts to 295 nm by incorporation in the sol-gel matrix. The very weak shoulder observed in the solution spectrum at ∼330 nm,

corresponding to d-d transitions, is probably overlapped with the strong LMCT band when encapsulated. The shift to 295 nm by encapsulation is associated with charge transfer from the hydroxo ligand in the species [Ru(NH3)5OH]2+, an intermediate formed by the hydrolysis of the deprotonated species [Ru(NH3)5NH2]2+ (eq 4).26,30 This means that the coordination sphere of Ru(III) changes, even at the low temperature used in method II, although an early decomposition product is obtained. In basic solution, the deprotonation of the Ru complex (eq 2) is evidenced by the broadband with a maximum at ∼400 nm, assigned to the LMCT from NH2- in the species ([Ru(NH3)5NH2]2+), which confers the solution a deep yellow color.25,30 Encapsulation of the Ru complex at 60 °C (method I) results in a clear reduction of the LMCT band to a shoulder of a new very strong band centered at 339 nm, and in the appearance of a weak Ru-brown related band at lower energy (∼463 nm). The band at 339 nm may be interpreted as (i) a Ru-brown related band; (ii) a Cl f Ru3+ CT typical of [Ru(NH3)5Cl]2+, an intermediate that can be formed by ligand exchange in the sol-gel medium;30,31 (iii) an increased d-d band of the Ru complex, as a consequence of a higher distortion of its octahedral geometry due to confinement effects and/or electrostatic interactions with siloxy (SiO-) groups of the silica matrix.27,30,31 Either way, it becomes clear that encapsulation

3992


Figure 4. DRIFT spectra of solid state [Ru(NH3)6]Cl2 (dashed), [Ru(NH3)6]Cl3 (gray), and Ru-red (black), normalized to the maximum.

of the Ru complex by method I results in an extended decomposition compared to method II. The spectra of fresh acid and basic aqueous solutions of [Ru(NH3)6]Cl2 are similar (Figure 3, lower): a very strong LMCT band at 263 nm plus a shoulder at ∼347 nm, assigned to d-d transitions. Upon encapsulation by method II, a new band located at 333 nm emerges, whose assignment may be discussed as above (339 nm band of the Ru(III) complex). A very weak feature in the region of 480 nm is indicative of an extremely low proportion of Ru-brown. When encapsulated by method I, the LMCT band is lost, and a very strong doublet appears with apparent maxima at 347 and 367 nm. This splitting can result from a distortion of the Ru complex, as proposed above, in addition to a higher contribution of the Ru-brown related band. The strong Ru-brown band at 481 nm clearly evidences an extended conversion to this trimer. The highest yield in Ru-brown while encapsulating the Ru(II) complex by method I accounts for the color evolution throughout the sol-gel process. This point will be readdressed in section 3.4, where a quantitative analysis of some spectra in Figure 3 will be made. The infrared spectra of solid state Ru(II) and Ru(III) hexaammine complexes have been well characterized in the past, assuming a skeletal Oh symmetry lowered by the crystal site.17,33,53–55 In the diffuse reflectance spectrum of [Ru(NH3)6]Cl2 (Figure 4, dashed), the δsNH3 and FNH3 modes are the dominant bands, at 1220 and 769 cm-1, respectively. The degenerate δsNH3 band is not split but has two shoulders at 1257 and 1319 cm-1. The νasNH3 and νsNH3 modes appear partially overlapped at 3309/3232 cm-1 and the δasNH3 mode as a medium intensity band at 1612 cm-1. The νRu-N modes, not observed in previous reports,53 are clear at 538 and 457 cm-1. In the spectrum of [Ru(NH3)6]Cl3 (Figure 4, gray), the strongest bands are also the δsNH3 (at 1319 cm-1, with two shoulders at 1362 and 1340 cm-1) and FNH3 (at 789 cm-1) modes. The bands at 3209/3199, 1620, and 465 cm-1 are assigned to the νasNH3/νsNH3, δasNH3, and νRu-N modes, respectively. In agreement with previous observations,53 the vibrational modes of the NH3 ligands are quite sensitive to the metal charge: a higher metal oxidation state implies stronger Ru-N bonds and thus weaker N-H bonds. The infrared and resonance Raman spectra of Ru-red have been analyzed in close correlation with the structural proposals.33,40,51,56 The strongest bands in the DRIFT spectrum (Figure

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Figure 5. (A) DRIFT spectra of the control silica (black) and the samples [Ru(NH3)6]Cl2/SiO2 (dashed), [Ru(NH3)6]Cl3/SiO2 (dotted), and Ru-red/SiO2 (gray) prepared by method I. (B) Enhanced region (1275-1365 cm-1) of the same spectra. (C) Same as part B except using method II.

4, black) correspond to the νasNH3 and νsNH3 modes, welldefined at 3242 and 3167 cm-1, respectively, followed by the δsNH3 bands, at 1300 and 1047 cm-1. The δasNH3 mode appears at 1612 cm-1. The band at 812 cm-1 and the shoulder at 738 cm-1 are assigned to the FNH3 and νasRu-O-Ru modes, respectively, although some authors have proposed the opposite assignment.33 The weak bands at 550 and 461 cm-1 are assigned to the νRu-N modes. When these Ru complexes are encapsulated in the sol-gel matrix by method I, and despite the reasonably high load used for [Ru(NH3)6]Cl2 and [Ru(NH3)6]Cl3, the DRIFT spectra result very similar to that of the control silica (Figure 5A). The main bands at 1090, 950, and 800 cm-1 are assigned to the νasSi-O-Si, νSi-OH/νSi-O-, and νsSi-O-Si modes, respectively, the broadband centered at 3200 cm-1 to the νOH mode, and the weaker bands at 1630 and 1402 cm-1 to the δHOH mode of water and δsCH3 of residual solvent.57 In the region of the strong δsNH3 mode of [Ru(NH3)6]3+, expected at 1319 cm-1, there is no significant matrix absorption, and it is clear that this Ru complex band is absent (Figure 5B, dotted). Instead, a shoulder appears at 1300 cm-1, assigned to the δsNH3 mode of ruthenium trimers such as Ru-red or Rubrown.33 The equally strong rocking mode is not detected because it overlaps with the νsSi-O-Si of the matrix. Nevertheless, when encapsulating the same nominal amount of Ru complex by method II, the δsNH3 mode is visible at 1319 cm-1 (Figure 5C), indicating that at least part of the ammine ligands were kept, in line with the evidence from the UV-vis spectra. If the δsNH3 mode of encapsulated [Ru(NH3)6]2+ were present, it would be masked by the νasSi-O-S band of the matrix. However, the DRIFT spectrum of the Ru complex encapsulated by method I (Figure 5B, blue) exhibits a weak shoulder at 1300 cm-1, confirming the partial transformation into Ru-red or Ru-brown. Encapsulation by method II results in a spectrum (Figure 5C) with a second shoulder at ∼1319 cm-1, characteristic of [Ru(NH3)6]3+, confirming that full oxidation is delayed but not entirely inhibited. These results corroborate the higher instability of the Ru(II) complex and that the decomposition occurs through [Ru(NH3)6]3+. The DRIFT spectrum of encapsulated Ru-red does not provide information due to its low solubility in the sol-gel mixture.


Figure 6. UV diffuse reflectance spectra (upper) of the purine bases encapsulated in silica matrixes by methods I (gray) and II (black), normalized to the maximum, and subtracted for the very weak spectrum of the control silica; UV absorption spectrum (lower) of the purine bases in aqueous solution.

Combining the information drawn from the infrared and UV-vis spectra of the encapsulated Ru complexes, we conclude that it is possible to control their oxidation extent through the encapsulation conditions, and even achieve incorporation preserving their chemical structure. The temperature proved to be a more influential parameter than the catalysis conditions. 3.3. Effects of Encapsulation on the DNA Purine Bases. The UV absorption spectra of the purine bases in aqueous solution are compared to their diffuse reflectance spectra when encapsulated in the silica matrixes in Figure 6. The spectrum of Ade in solution apparently consists of just one band centered at 262 nm that remains almost at the same position (260 nm) upon encapsulation. By spectral deconvolution into a sum of Gaussians (by a nonlinear least-squares fitting method), two components of equivalent intensities were retrieved, at 251 and 267 nm, assigned to the first π-π* transitions.58 No n-π* transitions could be recovered by deconvolution. Very similar results were obtained for encap-

J. Phys. Chem. B, Vol. 114, No. 11, 2010 3993 sulated Ade by methods I and II. These spectral deconvolutions are available as Supporting Information (Figures S1-S3). For Gua in solution, there are apparently two bands, but the best fit was achieved with three components, at 248 (70%), 272 (8%), and 283 (12%) nm (Figure S4 and Table S1 in the Supporting Information). On the basis of the relative intensities, we tentatively assign them to π-π*, n-π*, and π-π* transitions, respectively, given the much lower oscillator strengths of the n-π* transitions.59,60 It is known that hydrogen bonding of electron lone pairs commonly produces a substantial blue shift of n-π* transitions that may overlap with the stronger π-π* bands.60,61 By encapsulation within the silica network, the spectrum of Gua broadens in the high wavelength region, and the relative intensities of the two bands become more similar, particularly for the synthesis by method II. The spectral deconvolution retrieved four components: at 243 (34%), 280 (60%), 310 (6%), and 314 (less than 1%) nm by method I and at 245 (34%), 280 (51%), 306 (12%), and 312 (2%) nm by method II (Figures S5 and S6 and Table S1 in the Supporting Information). The two weaker components in both cases are assigned to n-π* transitions. Encapsulation results in a red shift of these transitions, which suggests a lesser involvement of the electron lone pairs in hydrogen bonds with the silica network than with the solvent. Besides, their relative intensity increases for encapsulation by method II, which points to a larger population of lone electron pairs not involved in H bonds. The DRIFT spectra of the solid phase DNA purine bases have been analyzed in detail elsewhere, as well as the effects of encapsulation in a silica matrix following method I.48 The influence of the encapsulation procedure is illustrated in Figure 7 for the more informative spectral region (1500-1800 cm-1), corresponding to the δNH2/νCO modes. The two bands in the spectrum of Ade crystals (at 1672 and 1604 cm-1, assigned to the δNH2 and δNH2/νC6C5 modes, respectively) are resolved in three components for the samples prepared by method I. The shoulder at 1606 cm-1, assigned only to the νC6C5 mode, was not affected by specific interactions. The stronger bands, at 1689 and 1651 cm-1, were assigned to NH2 groups with nonequivalent hydrogen bonding.48 Since the δNH2 bands are shifted to higher wavenumbers with respect to crystalline Ade, it is clear that the amine groups are more strongly bonded within the silica network. Encapsulation by method II does not affect the νC6C5 band either (1601 cm-1). However, only one strong δNH2 band appears at 1653 cm-1,

Figure 7. Region between 1500 and 1800 cm-1 of the DRIFT spectra of (A) pure Ade (black) and encapsulated in the silica matrix by methods I (gray) and II (dashed); (B) same as part A except for Gua. The spectra were normalized to the maximum.

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Figure 8. UV-vis diffuse reflectance spectra of (A) Ade/SiO2 (black), Ru-red/SiO2 (gray), and Ru-red/Ade/SiO2 (dashed), encapsulated by method I; (B) same as part A except encapsulated by method II; (C) Ade/SiO2 (black), [Ru(NH3)6]Cl3/SiO2 (gray), and [Ru(NH3)6]Cl3/Ade/SiO2 (dashed), encapsulated by method I; (D) same as part C except encapsulated by method II; (E) Ade/SiO2 (black), [Ru(NH3)6]Cl2/SiO2 (gray), and [Ru(NH3)6]Cl2/ Ade/SiO2 (dashed), encapsulated by method I; (F) same as part E except encapsulated by method II. The spectra were normalized to the maximum. Insets: enhanced spectral regions of interest for the Ru complex, normalized at ∼460 nm.

with the high wavenumber component reduced to a shoulder (∼1685 cm-1). This points to NH2 groups more homogeneously H bonded to the matrix than using method I, but without establishing the stronger interactions, which may be due to a more efficient condensation. For Gua encapsulated by method I, there is an inversion of the relative intensities of the stronger bands with respect to Gua crystals, which leads to assigning the band at 1674 cm-1 to the νCO mode and the component at 1695 cm-1 to the δNH2 mode, both groups more involved in hydrogen bonds than in pure Gua, where they appear at 1703 and 1674 cm-1, respectively.48 The weak band at 1639 cm-1 in the spectrum of crystalline Gua, assigned to the νC2N3 and νC4C5 modes, gains relative intensity and shifts to 1633 cm-1 when it is encapsulated by method I, probably due to a contribution from the δNH2 mode of amine groups with weaker hydrogen bonds. The spectral pattern is less altered when Gua is encapsulated following method II, the bands being comparable to those observed in crystalline Gua. Thus, the specific interactions between the CO and/or NH2 groups of the purine and the silica matrix are weaker when method II is followed. This is certainly related to a lower content in SiOH/SiO- groups, due to a more efficient condensation promoted by the more basic medium. Nevertheless, the band at 1635 cm-1 (νC2N3 and νC4C5) is relatively stronger, as

for Gua encapsulated by method I. This could be due to a contribution of more free NH2 groups within the matrix, or just to the δHOH mode of residual water. The bands at 1564 and 1554 cm-1 in Gua crystals (δCN9H modes of the imidazole ring) are not affected by either method of encapsulation. 3.4. Interactions between Coencapsulated Ru Complexes and Purines. For the different Ade/Ru complex pairs, the UV-vis diffuse reflectance spectra (Figure 8) are dominated by the π-π* bands of Ade, at ∼260 nm, characterized by very high absorption coefficients. They do not show relevant modifications, independently of the complex and coencapsulation method, suggesting that any interactions between Ade and the Ru complexes do not involve the π system of the purine. For the Gua/Ru complex pairs (Figure 9), there is no predominance of the purine nor the complex bands, since the absorption coefficients are of the same order of magnitude. Coencapsulation by method I induced the most significant modifications in the spectra, particularly for complexes [Ru(NH3)6]3+ and [Ru(NH3)6]2+ (Figures 8C,E and 9C,E). For this reason, a quantitative analysis of these spectra was carried out by band deconvolution into a sum of Gaussian components, using a nonlinear least-squares fitting method. The results are summarized in Table 4.



Figure 9. UV-vis diffuse reflectance spectra of (A) Gua/SiO2 (black), Ru-red/SiO2 (gray), and Ru-red/Gua/SiO2 (dashed), encapsulated by method I; (B) same as part A except encapsulated by method II; (C) Gua/SiO2 (black), [Ru(NH3)6]Cl3/SiO2 (gray), and [Ru(NH3)6]Cl3/Gua/SiO2 (dashed), encapsulated by method I; (D) same as part C except encapsulated by method II; (E) Gua/SiO2 (black), [Ru(NH3)6]Cl2/SiO2 (gray), and [Ru(NH3)6]Cl2/ Gua/SiO2 (dashed), encapsulated by method I; (F) same as part E except encapsulated by method II. The spectra were normalized to the maximum. Inset: enhanced spectral region of interest for the Ru complex, normalized at ∼460 nm.

As expected, the positions and relative areas of Ade components when coencapsulated with [Ru(NH3)6]3+ or [Ru(NH3)6]2+ compare well with those obtained for Ade encapsulated by the same method, confirming the noninvolvement of the purine π system. The same may be said for the π-π* bands of Gua as a whole. The n-π* component does not shift with respect to Gua within silica, which points to no preferential interactions with the Ru complex. However, its relative intensity increases when it is coencapsulated with [Ru(NH3)6]2+, which is indicative of an even higher population of free electron lone pairs. The spectral deconvolution of encapsulated [Ru(NH3)6]3+ allowed retrieving, in addition to the bands already assigned in Table 3, a small component at 300 nm related to the hydroxo decomposition intermediate, an important one at 365 nm assigned to distorted Ru trimers or further decomposition products,41 and a residual band at 577 nm related to Ru-red. Thus, a small amount of the Ru complex coexists, within the silica matrix, with different intermediates and Ru-brown. Coencapsulated Ade has a remarkable inhibiting effect on this oxidation process, since the main component becomes the LMCT of unreacted [Ru(NH3)6]3+ (that increases from 10 to 41%), and the relative area of the Ru-brown band reduces from 21 to 4%. Gua has the opposite effect: there is no residual

unreacted Ru complex, and the relative amount of the intermediate [Ru(NH3)5OH]2+ increases significantly (from 11 to 23%), as if it were stabilized by the purine. The recovered components of the encapsulated [Ru(NH3)6]2+ show that only the decomposition products remain encapsulated in the dry silica, mostly the intermediate with a hydroxo ligand (band at 299 nm, 22%) and Ru-brown (479 nm, 31%). Besides, a small band at 604 nm may be assigned to the dimers from Ru-red decomposition, such as [(NH3)5RuORu(NH3)5]5+ or Ru360.42,62 Coencapsulation of Ade results in a drastic reduction in the yield of Ru trimers. However, the purine does not inhibit the first steps of oxidation, since there is no unreacted complex left nor [Ru(NH3)6]3+, and the predominant intermediate has a hydroxo ligand. Gua has again an opposite effect, as the predominant species within the matrix are Ru trimers and dimers. Although not so evident in the case of Ru-red, due to its lower content, coencapsulation with Ade by method I results in the same type of inhibition, as shown by a higher proportion of the Ru complex in its initial form (in the inset in Figure 8A, it is clear that there is a significant absorption at ∼530 nm, without a higher contribution from the dimer band at ∼600 nm). Coencapsulation of Ru-red with Gua results in a larger contribu-

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TABLE 4: Results of the Band Deconvolution of the DRUV Spectra of the Purines and the Ru(II) and Ru(III) Complexes Encapsulated Separately and Coencapsulated, Following Method I assignments

π-π* (purine)

LMCT (NH3 f Ru3+)

d-da

eg * r eg (trimers)a

e g* r e u * (Ru-brown)

eg* r e u * (Ru-red)

300 11

335 23

365 27

458 21

577 8

262 62b

301 10c

337 23c

363 13c

430 4c

526 9c

277 45b

299 23c

336 19c

362 28c

450 19c

577 11c

299 22

337 3

374 33

479 31

559 2

604 9

337 26c

365 26c

460 11c

337 5c

369 41c

472 34c

567

Interactions between DNA Purines and Ruthenium Ammine

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