Kinetico-mechanistic Studies on the Substitution Reactivity on the

Article pubs.acs.org/IC

Kinetico-mechanistic Studies on the Substitution Reactivity on the {RuII(bpy)2} Core with Nucleosides and Nucleotides at Physiological pH Marta Vázquez and Manuel Martínez* Departament de Química Inorgànica i Orgànica, Secció de Química Inorgànica, Universitat de Barcelona, Martí i Franquès 1-11, E-08028 Barcelona, Spain S Supporting Information *

ABSTRACT: The kinetico-mechanistic study of the substitution reactions of the aquo ligands in cis-[Ru(bpy)2(H2O)2]2+ by different nucleotides and nucleosides has been conducted at pH close to the physiological value. The concentration dependence and thermal and pressure activation parameters have been measured to ascertain the activation via which reactions take place. Substitution processes are found associatively activated for nitrogen-bonded nucleosides or nucleotides, with outer-sphere hydrogen-bonded aggregates being determinant. For reactions leading to oxygen-bonded nucleotides, the process is clearly dissociatively activated. A selectively induced lability of the inert {RuII(bpy)2} core is observed on the formation of nitrogen(amide)-bonded complexes at relatively low pH values, which might be relevant for the effective intercalation of designed, ruthenium(II)-bonded, aromatic rings.

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coordination to biomolecules,14 some redox processes leading to ROS,15 and the use of intercalating molecules.16−18 The formal octahedral configuration (t2g6) is associated with an inert character,19−21 even when oxidized to ruthenium(III) (t2g5), which seems to be a rather good feature (see above), especially when the substitution reactivity is still occurring on a reasonable time scale. Furthermore, for some of the ruthenium(II) and ruthenium(III) complexes found to be biologically relevant, formation of the actually active ruthenium species is found to occur via initial substitution reactions; nevertheless, their speciation has not been systematically studied.22,23 We have recently been involved in the substitution reactivity of inert cobalt(III) complexes, with encapsulating (N)4 macrocycles and two aqua ligands in the cis position at physiological pH, with nucleotides and nucleosides.10,11,24 Our results have shown that speciation of these complexes in such a medium is a crucial point that has to be considered comprehensively (including the presence of hydroxo-bridged species); furthermore, tuning of the inertness can be easily achieved. The use of equivalent ruthenium(II) complexes for this type of reactivity represents an obvious and interesting aim, but for ruthenium(II) complexes, the presence of {HC−NH} units on the ligand is undesirable in aqueous solution at pH values close to 7, which produces ligand oxidation.25 Furthermore, the cavity size of the {Me2(μ-ET)cyclen} crossbridged ligand, already used for cobalt(III), appears also to be too small for stabilization of a second-row transition-metal(II)

INTRODUCTION The studies on medicinal inorganic chemistry are dominated by cis-[Pt(NH3)2Cl2], due to both the therapeutic results obtained, despite well-known side effects of its use, and the fundamental aspects involved in its biologically relevant reactivity.1,2 The involvement of substitution processes, including a necessary solvolysis to produce the active diaqua species, has been established as crucial, including its aqua/hydroxo speciation at physiological pH.3,4 In this respect, some detailed reports about the involvement of hydroxo-bridged species have also appeared,5 thus indicating a very complex solution chemistry of the species in the relevant reaction medium. Other metal complexes involved in biologically relevant processes6 also require initial substitution reactions in order to attain, or release, the active species or ligand. It is clear that the degree of inertness of the metal complexes involved in such processes has to be relatively high if they have to reach their expected target, for both interaction and release purposes,7 and avoid leaching to the medium.8 Furthermore, the use of complexes that could behave as dead ends, due to excessive inertness, also has to be avoided.9 This is especially relevant when the appearance of hydroxo-bridged species is inherent with the medium used.10,11 The use of ruthenium(II) complexes has been shown to be a very good platinum(II) alternative, as indicated for their good results in clinical trials and their lesser undesired side effects, somehow associated with the chemical and redox similarity of ruthenium(II) to the lighter iron(II).12 Even though the mode of action of platinum(II) complexes is very well established,13 the relevant activity of ruthenium(II) complexes has been associated with a variety of processes. These include © XXXX American Chemical Society

Received: April 21, 2016

A

DOI: 10.1021/acs.inorgchem.6b01003 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

in this work might be influenced by light, preliminary photostability of the aqua complex was conducted. UV−vis monitoring of the samples of the ruthenium complex at 1 × 10−4 M concentration, at the pH values used in the study, indicates that only very small changes occur upon standing for 5 h in daylight. These changes neatly reverse in the dark after 5 h and are completely absent when the spectrophotometers are kept covered. The complex solution was thus protected from light for the full set of studies conducted (Figure S1). The pK a values of the complex were determined spectrophotometrically to ascertain those reported in the literature (pKa1 = 8.9; pKa2 > 11).42 The values determined agree with what was expected; the high values being due to both the oxidation state of the metal center and it belonging to the second transition series. 43,44 No predominance of deprotonated (even monohydroxo) species occurs in the conditions used in our study. No polymerization to form hydroxo-bridged complexes is expected to be relevant, as was decisively found for equivalent studies on cobalt(III) complexes with tetradentate N-donor ligands.10,45,46 Similarly, baseconjugated substitution pathways are expected to be absent because of the lack of any NH groups in the inert skeleton.19,20,47 Nevertheless, at pH > 7.5, some residual presence of cis-[Ru(bpy)2(H2O)(OH)]+ might be important because of both the decrease of the positive charge of the complex and the presence of a better ligand in the ruthenium coordination sphere. Chloride. As for some cobalt(III) systems studied with the same aim as that in the present work,10,11,24 the reaction of the complex with chlorides, which may be relevant in in vivo fluids, has been conducted.3,48 Contrary to what had been observed for the cobalt(III) complexes of the cyclen family, in the present study some water by chloride substitution, under pseudo-first-order conditions, occurs in the 3−6 h time scale at room temperature. The spectral changes obtained indicate, nevertheless, a rather small displacement of the equilibria involved, even at a very large excesses of [Cl−] [200−1000-fold that of the ruthenium(II) complex; Figure 1]. This reactivity has been consequently considered irrelevant in the context of substitution by the other ligands studied. In all cases, a decrease of the rate with increasing pH is observed (Figure S2), which is in line with the increasing presence of monohydroxo species with increasing pH, leading to a less favorable {+}/{−} outer-

center. The use of polypyridylruthenium(II) complexes seems thus to be a good alternative, given its general importance as anticancer compounds26 and the well-established preparative procedures of aqueous solution stable complexes.27 Despite these facts, no definite knowledge of redox-free substitution mechanisms occurring on ruthenium complexes is available so far.19,28 The associativeness/dissociativeness of the substitution activation processes on the complexes has been found to be dependent on a large number of features. As a general rule, ruthenium(II) is much more labile than ruthenium(III), and while ruthenium(III) shows clear associative activation for water exchange, ruthenium(II) presents less associative behavior.29 Nevertheless, some dissociatively activated substitution reactivity has also been reported for complexes with strong trans-labilizing donors or able to react via base-conjugated pathways.29,30 With this background, we report here the kineticomechanistic studies of the reaction of cis-[Ru(bpy)2(H2O)2]2+ with the nucleosides and nucleotides indicated, with their pKa literature values,31−35 in Scheme 1. The results are also Scheme 1

compared with substitution by chloride and inorganic phosphate. The data obtained have also been checked for the absence of hydroxo-bridged dinuclear species, in order to avoid the presence of extremely inert complexes. No relevant photoactivity has been observed, but the data collected at variable temperature and pressure indicate that an associative/ dissociative activation tuning exists depending on the characteristics of the entering ligands. Interestingly, trans labilization of the bpy ligand is observed at the highest acidities used when the reaction is carried out with deprotonable uridine and thymidine ligands.

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RESULTS AND DISCUSSION The photochemistry of ruthenium complexes is a clear subject of interest.36 In fact, we have already been involved in studies on this matter.37 Given the importance that photodynamic therapy is attaining38−41 and that the reactivity studies intended

Figure 1. Comparison of the spectra of cis-[Ru(bpy)2(H2O)2]2+ (red) and cis-[Ru(bpy)2Cl2] (black) with that obtained after the reaction of diaqua species with chloride (blue) at 25 °C, [Cl−] = 0.1 M, [Ru] = 1 × 10−4 M, pH = 7.0 (HEPES), and I = 1.0 NaClO4. B


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Figure 2. (a) Time-resolved UV−vis spectral changes observed during the reaction of cis-[Ru(bpy)2(H2O)2]2+ with H2PO4−/HPO42− at 25 °C, [Ru] = 1 × 10−4 M, [H2PO4−/HPO42−] = 0.06 M, pH = 7.0 (HEPES), and I = 1.0 NaClO4. (b) Plot of the values of 1kobs obtained versus [phosphate] for the reaction indicated at different pH values.

Table 1. Summary of the Kinetics and Thermal and Pressure Activation Parameters Determined for the Systems Indicated at Different pH Values at 25 °C, I = 1.0 NaClO4 (n.d. = Not Determined) ligand H2PO4−/HPO42−

kon/s−1, koff/s−1

pH 6.0−8.0 (average)

−3 a

kon′ = 1.4 × 10 koff = 7.2 × 10−4 1 kon = 6.8 × 10−4 1 koff = 1.8 × 10−4 1 kon = 1.5 × 10−3 2 k = 1.1 × 10−4 1 kon = 5.0 × 10−3 2 k = 1.2 × 10−4 1 kon′ = 4.1 × 10−3 a 1 koff = 2.9 × 10−4 1 kon′ = 4.7 × 10−3 a 1 koff = 4.1 × 10−4 1 kon = 3.3 × 10−3 2 kon = 6.0 × 10−5 1 kon = 1.1 × 10−3 2 kon = 1.4 × 10−4 1

KOS/M−1

ΔH⧧/kJ mol−1

ΔS⧧/J K−1 mol−1

ΔV⧧/cm3 mol−1

non-limiting

n.d. n.d. 116 ± 4b 102 ± 1b n.d. n.d. 54 ± 4c 98 ± 5c 59 ± 5e 123 ± 8e n.d. n.d. n.d. n.d. 88 ± 4 43 ± 1

n.d. n.d. 84 ± 12b 20 ± 2b n.d. n.d. −116 ± 13c 5 ± 18c −96 ± 16e 96 ± 29e n.d. n.d. n.d. n.d. −13 ± 15 −179 ± 3

n.d. n.d. n.d. n.d. n.d. n.d. −26 ± 4c,d ∼0c,d n.d. n.d. n.d. n.d. n.d. n.d. −30 ± 3f −20 ± 3f

1

5′-CMP−/5′-CMP2−

6.0−8.0 (average)

5′-TMP−/5′-TMP2−

6.0 7.0−8.0 (average)

cytidine

6.0−7.5 (average) 8.0

thymidine

6.5 8.0

a

22 ± 7 17 ± 7 20 ± 6 non-limiting non-limiting 25 20 22 38

± ± ± ±

3 7 7 4

In M−1 s−1, non-limiting behavior. bpH = 6.5. cpH = 8.0. dAt 15 °C and [5′-TMP] = 0.1 M. epH = 6.5. fpH = 7.0, 20 °C, and [thymidine] = 0.1 M.

pH values (pKa2 = 7.2).50 Probably the increasing amounts of monocationic monohydroxoruthenium(II) species compensates this effect, with k′on being determined as the konKOS product from the general rate law indicated in eq 1, with the corresponding general mechanism.

sphere approach of the reactants ({2+}/{−} for the diaqua species). Phosphate. As a followup, substitution of the aqua ligands on the complex by inorganic phosphate has been pursued to determine the possible conditions for substitution by nucleotides. Figure 2a shows the time-resolved UV−vis spectral changes observed during the process; parallel 31P NMR spectral monitoring in equivalent conditions was also conducted. The latter indicates the sole formation of mono-η1-O-PO3 species as the substituted compound (signal at ca. 12 ppm lower field from the free ligand).49,50 Effectively, UV−vis spectral changes agree with a single substitution step (1kobs) when analyzed with Specf it51 or ReactLab52 software. The phosphate concentration dependence of the pseudo-first-order observed rate constants is linear, with a clear intercept (Figure 2b). This indicates that the process is in equilibrium under the conditions used for the study. From the kinetic data collected in Table 1, a value of the formation equilibrium constant of ca. 2.0 is derived, indicating only a small presence of the phosphato complexes under these conditions. This value is similar to that estimated from Figure S2 for the chloride substitution process. From the plot in Figure 2b, no dependence of the values of 1kobs on the pH is evident, despite the increasing amounts of HPO42− at higher

5′-CMP. After the conditions were set with inorganic phosphate, the reactivity of the complexes with nucleotides was studied. Given our previous knowledge of the distinct reactivity patterns of 5′-CMP and 5′-TMP, these ligand molecules were used (Scheme 1).11,24 The reactivity with 5′-CMP, followed by UV−vis spectroscopy under pseudo-first-order conditions, agreed with the occurrence of a single step with a bathochromic shift in the position of the maxima. From these changes, the C


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Inorganic Chemistry

inorganic phosphate (Keq = konKOS/koff ≈ 75 M−1) indicates a thermodynamic preference for the nucleotide ligand. 5′-TMP. The substitution of aqua ligands on these ruthenium(II) species by 5′-TMP was also studied in view of the distinct behavior observed for tetraaminediaquacobalt(III) complexes,11,24 with the difference with respect to 5′-CMP being related to the presence of a deprotonable R2NH group in the thymine moiety of the nucleotide.32 The time-resolved UV−vis spectral changes observed (Figure 4a) agree with the actuation of a two-step consecutive process that can be easily resolved by the standard software; the faster step (1kobs) is found to be [5′-TMP]-dependent, while the second (2kobs), slower step, does not show any dependence on the entering ligand concentration (Figure 4b). 31P NMR parallel monitoring was also conducted to ascertain the nature of the species involved in the two steps observed. Under the low [RuII] conditions of the study, the nature of the product after the first step cannot be unequivocally determined because of the time needed for recording a reasonable 31P NMR spectrum. Only the product after the second step, showing a single signal at 13 ppm, could be characterized, indicating that the full process corresponds to the formation of a mono-η1-O-5′-TMP species.50 By comparison with similar systems studied, the first reaction must thus correspond to the formation of a monoη1-N-5′-TMP complex, with the concurrent deprotonation of the R2NH group of the thymidine unit.32 This complex must then evolve to the final oxygen-bound complex (or to a nitrogen-bound ⇄ oxygen-bound equilibrium mixture); obviously, this second step must be 5′-TMP concentrationindependent, as effectively observed (Scheme 2). It is

values of 1kobs could be determined, as indicated in the Experimental Section, and from their limiting [5′-CMP] dependence (see eq 1), the values 1kon, KOS, and 1koff could be calculated (Figure 3). The equilibrium nature of the process

Figure 3. Plot of the values of 1kobs obtained for the reaction of cis[Ru(bpy)2(H2O)2]2+ with 5′-CMP at 25 °C, [Ru] = 1 × 10−4 M, pH = 8.0 (HEPES), and I = 1.0 NaClO4.

was also confirmed by the increase in the spectral changes with the value of [5′-CMP]. Table 1 collects the data derived, as well as the thermal activation parameters determined at pH = 6.5. As for the inorganic phosphate reactivity, 31P NMR monitoring of the samples showed the sole appearance of a signal at 13 ppm, which agrees with the formation of a η1-O-5′-CMP form50 in a cis-[RuII(bpy)2(O-5′-CMP)(H2O)] species. As indicated in Table 1 (see also Table S1), neither the values of 1kon nor those of 1koff show significant trends with pH. Taking into account that anation (kon) occurs on the aqua ligand attached to the ruthenium(II) center and that aquation (koff) takes place on the fully deprotonated coordinated 5′CMP2− ligand at this pH range, a fairly dissociative activation is thus implied for both reactions.19 Effectively, the thermal activation parameters are indicative of dissociatively activated processes.20 Along the same line, the values determined for KOS are found surprisingly independent of the pH, despite the increasing amounts of 5′-CMP2− at higher pH (pKa3 = 6.0).31 The compensating changes in charge from the {Ru(H2O)2}2+/ {5′-CMP}− pair to {Ru(OH)(H2O)}+/{5′-CMP}2− with increasing pH could be held responsible for this fact, as for the phosphate substitution indicated above. The larger value of Keq for formation of the complex compared with that of the

Scheme 2

noticeable that the UV−vis spectral changes shown in Figure 4a include a feature not observed for the substitution of aqua ligands with 5′-CMP, i.e., an absorbance increase around 650 nm. Even though such changes cannot be interpreted with the

Figure 4. (a) Time-resolved UV−vis spectral changes observed during the reaction of cis-[Ru(bpy)2(H2O)2]2+ with 5′-TMP at 25 °C, [Ru] = 1 × 10−4 M, [5′-TMP] = 0.04 M, pH = 7.0 (HEPES), and I = 1.0 NaClO4. (b) Plot of the values of 1kobs and 2kobs obtained versus [5′-TMP] for the reaction indicated. D


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Inorganic Chemistry

of the OH− anion. The value of the formation equilibrium constant for this system (Keq = kon′/koff ≈ 15 M−1) indicates a less thermodynamically stable nucleoside complex compared with the equivalent nucleotide. The thermal activation parameters determined for the system (Table 1) indicate an associative activation for the 1kon′ reaction, while a dissociatively activation seems to operate for the reverse process. The fact that for this system kon′ includes a contribution of an outersphere association equilibrium constant (k′on = konKOS, eq 1) can explain this fact, apparently contrary to the microreversibility principle.57,58 An associative component has been added to the activation parameters determined for the secondorder kon′ rate constant. Thymidine. The studies were also conducted with the deprotonable thymidine nucleoside;33 in this case, a two-step consecutive process was observed in the time-resolved UV−vis spectral changes. As shown in Figure 5, the two pseudo-first-

data collected so far, they should be kept in mind for the studies on substitution by nucleosides (see below). These changes completely parallel the two steps observed and are more relevant at low pH. Nevertheless, they do not agree with a simple water by nucleotide substitution, while keeping the same local symmetry on the ruthenium(II) center.53,54 As seen in Figure 4b, the concentration dependence of 1kobs corresponds to the full rate law indicated in eq 1. Furthermore, as seen in Tables 1 and S1, no pH dependence is observed for this first step in the 7.0−8.0 range; its decrease at pH = 6.0 and 6.5 could be related to the pKa2 value (6.5)32 of 5′-TMP. Increasing amounts of the single protonated monoanionic phosphato species (5′-TMP−) should be present in the reaction medium compared with the less acidic 5′-CMP (equivalent pKa = 6.0),31 where no trend has been observed. This effect should also reflect the values of KOS, but this is not so because of the fact that the formation of a mono-η1-N-5′-TMP species does not derive from a simple ion-pair encounter complex. As a whole, this behavior points to the operation of an associatively activated process, as indicated by the thermal and pressure activation parameters determined at pH = 8.0 (Figure S3a), collected in Table 1.19,20 The value of ΔV⧧ is rather large when the partial molar volume of water (18 cm3 mol−1) is considered, but the fact that the system involves a rather large water cage can easily explain this fact, as was already found for similar CoIII systems studied.11,24 Clearly, the different nature of the final species is extremely relevant to the mechanism involved. While for 5′-CMP the formation of a mono-η1-O complex occurs via a dissociatively activated mechanism (see before), for substitution by 5′-TMP, an associative activation process is operative. A mono-η1-N complex is produced in more favorable terms (no equilibrium detected under the conditions used) than those for the mono-η1-O complex of 5′-CMP. Substitution by 5′-TMP on these complexes thus indicates that the presence of a deprotonable R2NH group produces enhanced substitution reactivity, with hydrogen-bonding interaction playing a determinant role,11 as well as for similar systems studied.32 For the second step observed (2kobs), the process is both pHand [5′-TMP]-independent, as expected for a nitrogen-tooxygen-bonding isomerization. Furthermore, the activation parameters determined and collected in Table 1 are, indeed, extremely different from those obtained for the first step (entry of the ligand). The high value of the activation enthalpy and practically null entropy and volume of activation are in line with those expected for the isomerization process.55,56 Cytidine. The study of the substitution processes by the nucleoside moieties of the above nucleotides was pursued to establish the differences when only N donors are present on the ligands. For the reaction with the nondeprotonable cytidine nucleoside, a single step is observed (1kobs) in the UV−vis spectral time-resolved changes, indicating the formation of a monosubstituted aqua species with a cytidine attached by the primary −NH2 amine. This behavior differs from that observed for previously studied cobalt(III) complexes, where the entry of two nucleoside units was observed. Under the studied conditions, the process is in equilibrium without significant formation of outer-sphere aggregates (i.e., koff ≠ 0 with (1 + KOS[ligand]) ≈ 1 in eq 1). The data collected in Table 1 indicate that the 1kon′ process is independent of the pH in the 6.0−8.0 range, as would be expected from the prevalence of the same charge for both reacting species in the range. Nevertheless, the reverse process (1koff) is slightly accelerated at pH = 8.0, in line with the increasing amounts in the reaction medium

Figure 5. Plots of the dependence on [thymidine] of kobs for the two steps observed on the time-resolved UV−vis spectral changes of the reaction of cis-[Ru(bpy)2(H2O)2]2+ with thymidine at different pH (HEPES) values and at 25 °C, [Ru] = 1 × 10−4 M, and I = 1.0 NaClO4.

order rate constants (1kobs and 2kobs) show a limiting nonequilibrium dependence on the concentration of thymidine; the derived kon and KOS values are collected in Tables 1 and S1 (Scheme3). No equilibrium is established under the conditions Scheme 3

used, thus indicating a definite preference for thymidine compared with cytidine. From the data collected, it is evident that 1kon shows a definite increase with decreasing pH in the 6.5−8.0 range studied, which is related to the dominant presence of the diaquo complex. A productive orientation59 by hydrogen bonding of the carbonyl groups on thymidine with the protons of the aqua ligands can be held responsible for this fact, as has already been proposed for the (CoIII{μ-ET)cyclen}) system with relatively acidic amine bonds.11 The thermal and pressure activation parameters determined for this step indicate an associative activation mechanism operating for the first water by thymidine substitution. The breaking of a number of E


Inorganic Chemistry hydrogen-bonding interactions, thus increasing partially the disorder of the system (ΔS⧧ not very negative), is in good agreement with this reasoning. For the second step, the trend obtained for 2kon with the pH is opposite, as indicated in Tables 1 and S1. In this case, there is no spectator aqua ligand involved in the process, and the increase in the pH simply enables abstraction of the thymidine proton of the ligand in the outer-sphere precursor. Again, the thermal and pressure activation parameters determined indicate an associative activation mechanism (Figure S3b), as found for all of the processes, leading to the ruthenium(II)-to-nitrogenbonded nucleotides or nucleosides studied. This is distinct from the processes leading to oxygen-bonded nucleotide analogues. As stated before (Figure 4a), Figure S4 shows an example of the interesting rather definite changes in the UV−vis spectrum in the 650 nm zone at pH < 7.0. These changes even become dominant at pH = 6.0, preventing a reproducible quantification of water by the thymidine substitution process. This is specifically a nitrogen-bonded thymidine effect, which confirms the initial nitrogen bonding of 5′-TMP, followed by oxygenbonding isomerization, proposed before. Given the distinct UV−vis spectral signature of the process, 1H NMR monitoring of the reaction obtained upon substitution by thymidine was conducted at pH = 6.0. The PRESAT 1H NMR spectra showed a set of rather wide signals at 25 °C, widening at 50 °C and narrowing at 10 °C (Figure S5a), indicating that fast movement is taking place at room temperature. Parallel electrochemical monitoring was also carried out to account for any possible oxidation to paramagnetic ruthenium(III) complexes. As seen in Figure S5b, no changes in the ruthenium(III)/ruthenium(II) potential are observed upon substitution, thus indicating that no ruthenium(III) species were formed upon complexation. It is clear that, upon RuII−NR2 coordination, a fluxional process takes place on the {RuII(bpy)2} core, especially at low pH, where the N donors of the bipyridine could be partially protonated upon decoordination (pKa = 4.4).60 In this way, such electronically induced47,61 dangling trans movement should be made easier, producing equilibrium amounts of putative lower-symmetry [Ru(η2-bpy)(η1-bpy)(N-thymidine)(H2O)]+ species (Scheme 4). In this respect, a complete set of

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CONCLUSIONS

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EXPERIMENTAL SECTION

The kinetico-mechanistic study of the substitution reactions of aquo ligands in cis-[RuII(bpy)2(H2O)2]2+ by different nucleotides and nucleosides has been conducted at pH close to the physiological value. The high pKa values of the complex prevent the formation of dead-end hydroxo-bridged species, which have been found very relevant for systems with other metals. While the disubstituted complex is only formed when nucleosides have a deprotonable amine group, i.e., thymidine or uridine, the monosubstituted complexes are obtained for 5′-CMP, 5′-TMP, and cytidine. Even in these cases, the processes appear to be in equilibrium, despite the high excesses of the entering ligand used. For all reactions, a thermodynamic preference for oxygenbonded nucleotides is clear, which includes a nitrogen-tooxygen-bonded isomerization for the substitution by 5′-TMP. In this respect, the substitution processes are found associatively activated whenever a nitrogen-bonded nucleoside or nucleotide is attained. The formation of well-oriented hydrogen-bonded outer-sphere aggregates, prior to aqua ligand substitution, can be held responsible for this fact. For the reactions leading to oxygen-bonded nucleotides, the process is dissociatively activated in all cases. An especially stricking feature of the processes studied relates to the selectively induced lability of the originally inert bipyridine ligand on the ruthenium(II) center at relatively low pH values. The formation of a nitrogen(amide)-bonded complex produces important labilization of the otherwise inert {RuII(bpy)2} core.

Compounds and Procedures. The ruthenium cis-[Ru(bpy)2(H2O)2](CF3SO3)2 complex has been prepared from cis[Ru(bpy)2Cl2], as described.27 The diaquo complex was prepared following the literature procedure64 with some modifications. The dichlorido complex was dissolved in water under a nitrogen atmosphere, and two equivalents of Ag(CF3SO3) were added in a slightly acidic medium; after filtering of the AgCl formed, the solution was taken to dryness. The UV−vis spectrum of the solid in water [λmax = 340 nm (8260 M−1 cm−1); λmax = 480 nm (9770 M−1 cm−1)] agrees with that reported. 2-(N-Morpholino)ethanesulfonic acid (MES) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) solutions were prepared to a 0.4 M concentration at I = 1.0 (NaClO4) by weighing the desired amounts of commercially available reactants. The final pH was adjusted with suitable HClO4 or NaOH solutions. In all cases, buffering was checked before and after the reactions studied. These stock solutions were used as solvents for all of the ligand solutions used in the study. 1 H and 31P NMR spectroscopy was carried out on a Bruker 400Crio instrument in water adjusted at the desired pH and with a D2O inset containing the corresponding reference, at Unitat d’RMN from the Centres Cientı ́fics i Tecnològics de la Universitat de Barcelona, and referenced externally to NaTMSP (1H) and H3PO4 (31P). 1H NMR spectra from the aqueous solutions were collected using a water PRESAT experiment on the same instrument. UV−vis spectra were collected using either a Cary 50 or a HP8453 instrument equipped with thermostated multicell transports. For the reactions carried out at varying pressure, an already described65−68 pillbox cell and pressurizing system were used, connected to a J&M TIDAS instrument. pH measurements were conducted on a Crison instrument using either fast-response or microsample-glass combined electrodes. Timeresolved UV−vis spectra were collected with the same instruments indicated above and exported to the relevant software packages indicated below.

Scheme 4

experiments was repeated using uridine as the entering nucleoside. Both the kinetics and UV−vis and 1H NMR spectra perfectly match those observed for thymidine, indicating the feasibility of the discussion above. This is an important finding given the fact that some of the ruthenium(II) complex bioactivity is related to possible intercalation of ruthenium-bonded aromatic ligands in DNA chains,12,62 especially relevant in the more acidic media found in tumors.63 F


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Electrochemistry experiments were carried out with a BioLogic SP150 instrument using a glassy carbon working electrode, a Ag/AgCl (saturated KCl) reference electrode, and platinum wire counter electrode using 0.1 M NaClO4 as the supporting electrolyte. All solutions were purged with nitrogen prior to the analyses, and the scan rate was set at 100 mV s−1. All potentials are given versus NHE, once corrected for the reference electrode used (E° Ag/AgCl/sat. KCl = +195 mV versus NHE). pKa determination was carried out by UV−vis spectroscopy titration on 1 × 10−4 M solutions of the ruthenium complex, taken to 0.01 M HClO4, by adding small aliquots of 0.1 M NaOH. The electronic spectra were recorded by using a Helma 661.202-UV All Quartz Immersion Probe connected to a Cary 50 instrument with optical fibers. The pKa values were calculated using the standard Specf it or ReactLab equilibrium software.51,52 Kinetics. Solutions of the different ligands involved in the kinetic runs were prepared in the corresponding 0.4 M buffer solutions at I = 1.0 described above. The solutions of the metal complex were prepared at much higher concentrations (at least 20−30-fold) in water; small aliquots of this stock solution were added to achieve the final conditions of the runs ([RuII] = 1 × 10−4 M, and [ligand] = 0.01−0.1 M). For all of the substitution processes, pseudo-first-order conditions were used. All of the time-resolved experiments were conducted according to the following setups: (i) For a nonbuffered medium, the desired aliquot of the stock ruthenium(II) complex solution was added to a solution at a chosen acidity, the pH was immediately registered, and further spectral changes were monitored. (ii) For experiments in buffered media, the desired aliquot of the stock ruthenium(II) complex solution was added to the chosen 0.4 M buffer solution, the pH was registered, and further spectral changes were monitored. All data were collected as full (300−750 nm) spectra and treated with the standard Specf it or ReactLab kinetic software;51,52 observed rate constants were obtained from the full changes of the spectra or alternatively at the wavelength where a maximum change was observed. The changes were fitted to the relevant A → B singleexponential equation when pseudo-first-order conditions were applied; for consecutive reactions with the same characteristics, an A → B → C double-exponential sequence was fitted. Table S2 collects all of the values obtained for kobs as a function of the different compounds and variables studied.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b01003. Values of the observed rate constants for the experiments described, its concentration dependence, and figures showing the spectral changes associated with some of these rate constants (PDF)

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Spanish Ministerio de Economiá y Competitividad/FEDER (Projects CTQ2015-65707C2-1 and CTQ2015-71211-REDT) is acknowledged. M.V. also acknowledges a FI-DGR grant from the Generalitat de Catalunya. G


Article

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Kinetico-mechanistic Studies on the Substitution Reactivity on the

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