Article pubs.acs.org/IC
Electrophilic Activation of Oxidized Sulfur Ligands and Implications for the Biological Activity of Ruthenium(II) Arene Anticancer Complexes Thamayanthy Sriskandakumar,† Shirin Behyan,† Abraha Habtemariam,‡ Peter J. Sadler,‡ and Pierre Kennepohl*,† †
The University of British Columbia, Department of Chemistry, Vancouver, BC V6T 1Z1, Canada University of Warwick, Department of Chemistry, Coventry CV4 7AL, United Kingdom
‡
S Supporting Information *
ABSTRACT: Surprisingly, the anticancer activity of half-sandwich Ru arene complexes [(η6-arene)Ru(en)Cl]+ appears to be promoted and not inhibited by binding to the intracellular thiol glutathione. Labilization of the RuS bond allowing DNA binding appeared to be initiated by oxygenation of the thiolate ligand, although oxidation by itself did not seem to weaken the RuS bond. In this study, we have investigated the solvation and acidic perturbations of mono (sulfenato) and bis (sulfinato) oxidized species of [(η6-arene)Ru(en) (SR)]+ complex in the presence of Brønsted and Lewis acids. Sulfur K-edge X-ray absorption spectroscopy together with density functional theory calculations show that solvation and acidic perturbation of sulfenato species produce a significant decrease in the S3p character of the RuS bond (Ru4dσ* ← S1s charge donation). Also there is a drastic fall in the overall ligand charge donation to the metal center in both sulfenato and sulfinato species. Our investigation clearly shows that mono oxidized sulfenato species are most susceptible to ligand exchange, hence providing a possible pathway for in vivo activation and biological activity.
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INTRODUCTION DNA is a common target for transition metal anticancer complexes, the most well-known example being Pt(II) complexes such as the clinical drug cisplatin. DNA is also thought to be the target for ruthenium(II) arene anticancer complexes such as [(η6-arene)Ru(en)Cl]+. Both cisplatin and the organo−Ru(II) chloride complexes have labile MCl bonds which become activated by aquation and bind strongly especially to guanine bases on DNA.1,2 However, cells contain millimolar concentrations of the thiol glutathione (GSH), which can potentially deactivate these complexes before they reach nuclear DNA on account of the formation of strong M S (thiolate) bonds.3,4 We were surprised to discover that the Ru(II) arene complexes still bind preferentially to DNA in the presence of large excesses of GSH, after formation of [(η6arene)Ru(en) (SG)]+ adducts. This appeared to occur by a mechanism involving oxidation (oxygenation) of the bound thiolate.5 We have therefore become interested in investigating the effect of thiolate S oxidation on the strengths of the RuS bonds and their reactivity.2,6,7 Specifically, oxygenation of the bound sulfur atom in RuII arene thiolato ([(η6-arene)Ru(en) (SR)]+) prodrugs may be an important step in their biological activation.8,9 It has been proposed that sequential oxidation and protonation of the thiolato ligand are required for efficient substitution of thiolato ligands by guanine N7 and DNA binding.8,10 Electronic structure studies of the thiolato, sulfenato, and sulfinato species have identified the mono© 2015 American Chemical Society
oxygenated (sulfenato) species as the most susceptible to MS bond dissociation upon protonation.9 Experimentally validated DFT calculations supported the assertion that protonation to the sulfenic acid complex should result in a large decrease in the RuS covalency, resulting in a weaker and more labile metal− sulfur bond. Importantly, the high concentration of glutathione in cells suggests that many, if not all, bioactive complexes of the form [(η6-arene)RuII(en)(X)]+ (X = Cl−, Br−, I−) would form the corresponding glutathione complexes [(η6-arene)Ru(en) (SG)]+ or similar protein-bound forms [(η6-arene)Ru(en)(P)]+ in vivo. Generally, sulfenates are too reactive to be isolated11 and readily disproportionate (to sulfinates + thiolates)12 unless coordinated to a transition metal. Similarly, their protonated analogs also pose synthetic challenges. Interestingly, the only reported Ru complex with protonated sulfenato on the oxygen atom (sulfenic acid) involves strong hydrogen bond stabilization of the OH group.13 Earlier studies8 have shown that protonation of ruthenium arene sulfenato complexes is possible in aqueous HCl solution at pH 2.11 (pKa of the protonated sulfenic acid is 3.61) in 16 h with partial hydrolysis. The reaction yields the reactive aqua adduct, [(η6-hmb)Ru(en)(H2O)]2+ (where hmb = hexamethylbenzene and en = ethylenediamine) and chlorido complex, [(η6-hmb)Ru(en) Received: November 9, 2015 Published: November 24, 2015 11574
DOI: 10.1021/acs.inorgchem.5b02493 Inorg. Chem. 2015, 54, 11574−11580
Article
Inorganic Chemistry
Lewis acid (ZnII) adducts are both more stable and inert,8,9 allowing for both experimental and computational studies on these systems. Importantly, data available for the sulfinato complexes provide a useful means to evaluate the computational models used to probe the more reactive sulfenato species.
(Cl)]+ supporting the idea that protonation of the sulfenato complex leads to rapid ligand dissociation. Experimental constraints in generating sufficiently high concentrations of the appropriate sulfenic acid complex had limited our ability to provide conclusive experimental evidence for decreased RuS bond covalency upon protonation of the sulfenato intermediates of importance in biological activity. As means of circumventing some of the inherent challenges, we have investigated the effect of Lewis acids on the nature of the RuS bond in a series of sulfenato and sulfinato complexes. Using a combination of spectroscopic and computational data, we explore the effect of electrophilic perturbations of terminal oxygen atoms in these oxidized ruthenium(II) arene complexes. Similar strategies have been adopted by others to investigate the role of redox-inactive metal ions (such as ZnII) serving as Lewis acids to evaluate their potential impact on the reactivity of biologically relevant metal complexes.14 Likewise, Kovacs, Solomon, and co-workers15,16 investigated the effect of direct interaction of ZnII on an iron− sulfenato complex.15 The effect of other Lewis acidic metal ions such as NaI,17,18 LiI,19 and PtII,20 with the metal−sulfenato oxygen has also been explored. This model for the biological mode of action of RuII arene complexes suggests that the mono-oxygenated sulfenato species are most susceptible to ligand exchange under acidic conditions.9 We thus seek to explore the effect of exogenous metal ions on the bonding in RuII arene complexes to provide insights into the effect of protonation on these species. Particular attention is paid to sulfenato species due to their importance in the proposed mode of action, although the effect on sulfinato species is also explored for comparative purposes. In addition, sulfinato species have been detected as coproducts with sulfenato species during reactions of parent RuII−arene chlorido complexes, [(η6-ar)Ru(en) (Cl)]+, with glutathione (GSH)21 and human albumin.22 This work explores the effect of Lewis acid complexation on complexes 1a ([(η6-p-cym)Ru(en) (SOiPr)]+) and 2a ([(η6-pcym)Ru(en) (SO2Ph)]+ as shown in Schemes 1 and 2,
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RESULTS AND DISCUSSION The effect of electrophilic perturbations of the terminal oxo of both sulfinato and sulfenato complexes on the nature of the RuS bond has been explored through a combination of spectroscopic and computational studies. Effect of Perturbations on Sulfinato Complexes. The effect of ZnII on the electronic structure of 2a (2aZn2+) is reflected in a significant difference in the S K-edge XAS spectra, as shown in Figure 1a. Most notably, the main feature in the spectrum of 2a, previously assigned to a combination of SCσ*/ SOσ* ← S1s transitions,9 broadens substantially into two features in 2aZn2+. Furthermore, the Ru4dσ* ← S1s transition is clearly resolved in 2a, whereas it becomes a poorly resolved shoulder in 2aZn2+ due to greater overlap with the SOσ* ← S1s feature. The Ru4dσ* ← S1s transition is particularly relevant as its intensity is directly proportional to the degree of covalency in the RuS bond through S3p donation into the Ru4dσ* orbital.23−25 Systematic peak fitting26,27 (see SI-2) was performed on the S K-edge XAS of 2a and 2aZn2+ to determine the degree of S3p character in each of the low-lying acceptor states (Table 1).28,29 The results suggest that even though the energies of the acceptor orbitals are highly perturbed in the presence of Zn2+, there are only minor changes in the S3p contributions to these orbitals. DFT results for 2a and 2aZn2+ are in reasonable agreement with the experimental S K-edge XAS (see Figure 1b and Table 1). Interactions between the sulfinato ligand and the metal center are localized in only one of the two Ru 4dσ* orbitals (the empty eg orbitals in these pseudo-Oh low-spin 4d6 complexes); the other Ru 4dσ* orbital (the LUMO with contributions primarily from the arene ligand) remains essentially unchanged in all cases investigated. We thus focus our analysis on the LUMO+1 orbital.9 The amount of S3p character in the Ru 4dσ* orbital (LUMO+1) changes only slightly in the presence of the Lewis acid. A more detailed breakdown of contributions to this orbital, however, indicates that the small changes to the S3p contribution conceal a much larger change in the overall−SO2R ligand contribution. The overall charge donation from the sulfinyl ligand decreases dramatically, which correlates with weakening of the SO bond (SI-1). The decreased charge donation from the sulfinato ligand is compensated by increased donation from the ethylenediamine ancillary ligand (Table 1) such that the charge on the already electron-poor metal center remains essentially unchanged. These results are consistent with the anticipated effects of Lewis acid perturbations on the nature of the Ru-SO2R bonding.9 Computational results were also obtained on the protonated model 2aH+, which could not be investigated experimentally. Overall results from protonation of a single terminal oxo in the sulfinato ligand generates qualitatively similar results to that observed upon Zn2+ binding (2aZn2+). Overall donation from the sulfinato ligand to the metal center is substantially decreased upon perturbation of a terminal oxo on the ligand. The NBO-derived RuS bond order is found to be 0.28 in 2a, but is not observed for either 2aH+ or 2aZn2+. The RuSσ* interaction is listed as a sulfur lone-pair with occupancy of 1.19 (2aaq), 1.35 (2aZn2+), and 1.32 (2aH+). Similar results have
Scheme 1. (a) Protonation8 and (b) Lewis Acid Activation of RuII Arene Sulfenato (1) Complexes, Where R = p-cym (1a) or hmb (1b) and R′ = iPr
Scheme 2. (a) Protonation and (b) Lewis Acid Activation of RuII Arene Sulfinato Complex 2, Where R = p-cym (2a) or hmb (2b) and R′ = Ph
respectively. Experimental investigations of the sulfenatoLewis acid adducts proved very challenging, and as a result, the Lewis acid effect of sulfenates have been investigated using in silico models. However, the corresponding series of sulfinato 11575
DOI: 10.1021/acs.inorgchem.5b02493 Inorg. Chem. 2015, 54, 11574−11580
Article
Inorganic Chemistry
Figure 1. (A) Normalized S K-edge XAS of complexes 2a and 2aZn2+ (B) XAS and simulated TDDFT of complex 2aZn2+.
Table 1. Experimentally Determined S3p Charactera and Calculated Charge Distribution of Acceptor (Ru4dσ* ← S3p) Orbital in Sulfinato Complexes % S3p in acceptor complex
XAS
DFT
SO2R (%)
2a 2aaq 2aZn2+ 2aH+
6.3 ± 1.3%
6.2 5.5 5.2 5.8
23.1 16.9 12.7 11.3
5.8 ± 1.5%
H/M(H2O)2 (%)
Ru (%)
ar (%)
en (%)
1.3 0.0
45.7 45.9 45.6 43.7
21.5 23.4 25.1 25.7
6.0 9.7 11.4 15.6
a DFT-calculated S3p contribution to the acceptor orbital, Ru4dσ* ← S3p, is determined from a Löwdin charge decomposition of the Kohn−Sham orbital. Zn2+ accompanied by two H2O molecules and bound to both oxygen of the sulfinato to complete the tetrahedral coordination sphere.
calculated Kohn−Sham orbitals upon solvation (2aaq), Zn2+ coordination (2aZn2+), and protonation (2aH+). This effect correlates with a weakening of the SO bond in each case. The indirect effect of this interaction upon the RuSσ* orbital is extremely small, reflecting the fact that contributions from the terminal oxo groups into this orbital are relatively small in the sulfinato complexes.9 Effect of Perturbations on Sulfenato Complex. The reasonable agreement between experimental and computational data obtained for the sulfinato complexes under differing environments, as well as previous success of our DFT results in the solid state,9 provides us with sufficient confidence that an in silico exploration of the effect of solvation (1aaq), protonation (1aH+), and Zn2+ perturbation (1aZn2+) is appropriate. In each of these cases, the overall geometric perturbation relative to the reference system (1a) is localized to atoms that are within 1−2 bonds from the terminal oxo of the sulfenato ligand. As observed in the sulfinato case, perturbations trigger elongation of the SO bond and concomitant (small) contraction of the RuS bond (SI-3). A similar trend is also observed in the Lewis acid adduct of FeIII−sulfenato complex studied by Kovacs and co-workers.15,16 Overall, the observed effect of electrophilic perturbation of the terminal oxo follows very similar trends to those observed in the sulfinato complexes, although some differences exist. As shown in Table 2, the perturbations to the sulfenato ligand have a greater impact on the S3p contributions in the RuSσ*, which reflects a more substantial decrease in donation into the metal center. This observation suggests that modulation of the terminal oxo group has a more direct influence on the metal−sulfur bond as compared to the sulfinato complexes. This is consistent with our previous observation that the RuSOR bond in sulfenato complexes involves the SO π bond, whereas this is not true for sulfinato
been observed by Shearer and co-workers for iron sulfinato complexes.30 Surprisingly, results obtained for 2a using in silico aqueous COSMO solvation (2aaq) already show a RuS bond that is intermediate between that of 2a and 2aH+. The overall effect can be seen in Figure 2, which graphically represents the calculated changes in the valence molecular orbital structure of these complexes. The most notable changes are seen in the orbitals with SOσ* character, which lower their energy relative to the rest of the
Figure 2. Simplified representation of relevant frontier molecular orbitals that change upon electrophilic perturbation of the terminal oxo groups on the sulfinato species (i.e., 2a → 2a-LA [LA = H2O, Zn2+ and H3O+ ]). The magnitude of the observed changes depends on the strength of the interaction (see SI). Significant observable changes occur in orbitals involved in SO and SRu bonding. 11576
DOI: 10.1021/acs.inorgchem.5b02493 Inorg. Chem. 2015, 54, 11574−11580
Article
Inorganic Chemistry Table 2. DFT-Calculated S3p Contributiona (Ru4dσ* ← S3p) and the Total Charge Distribution of Kohn-Sham Acceptor Orbital, Ru4dσ* of 1a, 1aZn2+, and 1aH+ Complexes complex
% S3p in acceptor
SOR (%)
1a 1aaq 1aZn2+ 1aH+
12.7 11.2 10.6 9.2
20.9 18.9 17.0 14.9
H/M(H2O)3 (%) Ru (%)
6.2 0.4
41.1 42.6 46.4 48.3
Ar (%)
en (%)
20.0 20.5 20.5 19.5
6.1 6.1 6.4 6.1
Our computational results also indicate that simple solvation (even within the very limited framework of the COSMO model) has a larger influence on 1a than 2a. This is confirmed by exploring the difference between the solid state spectrum of 1b and its spectrum in aqueous solution (Figure 4). Photodecomposition of samples in the X-ray beam was a significant problem, particularly at low pH, but rapid single scans (
