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Insights into Second-Sphere Effects on Redox Potentials, Spectroscopic Properties, and Superoxide Dismutase Activity of Manganese Complexes with Schiff-Base Ligands

Six Mn-Schiff base complexes, [Mn(X-salpn)]0/+ (salpn = 1,3-bis(sal-ic-ylidenamino)propane, X = H [1], 5-Cl [2], 2,5-F2 [3], 3,5-Cl2 [4], 5-NO2 [5], 3,5-(NO2)2 [6]), were synthesized and characterized in solution, and second-sphere effects on their electrochemical and spectroscopic properties were analyzed. The six complexes catalyze the dismutation of superoxide with catalytic rate constants in the range 0.65 to 1.54 × 106 M–1 s–1 obtained through the nitro blue tetrazolium photoreduction inhibition superoxide dismutases assay, in aqueous medium of pH 7.8. In solution, these compounds possess two labile solvent molecules in the axial positions favoring coordination of the highly nucleophilic O2•– to the metal center. Even complex 5, [Mn(5-(NO2)salpn) (OAc) (H2O)], with an axial acetate in the solid state, behaves as a 1:1 electrolyte in methanolic solution. Electron paramagnetic resonance and UV–vis monitoring of the reaction of [Mn(X-salpn)]0/+ with KO2 demonstrates that in diluted solutions these complexes behave as catalysts supporting several additions of excess O2•–, but at high complex concentrations (≥0.75 mM) catalyst self-inhibition occurs by the formation of a catalytically inactive dimer. The correlation of spectroscopic, electrochemical, and kinetics data suggest that second-sphere effects control the oxidation states of Mn involved in the O2•– dismutation cycle catalyzed by complexes 1–6 and modulate the strength of the Mn-substrate adduct for electron-transfer through an inner-sphere mechanism.


■ INTRODUCTION
The superoxide radical anion (O 2 •− ) is generated by the oneelectron reduction of O 2 during normal cellular metabolism and constitutes the primary source of the deleterious hydroxyl radical (HO • ) and hydrogen peroxide (H 2 O 2 ). Manganese superoxide dismutases (SODs) catalyze the conversion of intracellular O 2 •− into O 2 and H 2 O 2 and constitute one of the major antioxidant defense systems against this toxic metabolite. 1 The active site of these enzymes contains one Mn ion bound to three histidine ligands, one aspartate and water or hydroxide as the fifth ligand in a trigonal bipyramidal geometry, and carries out O 2 •− disproportionation through a redox process involving one-electron oxidation and reduction of the metal ion between Mn(II)/Mn(III) levels. 2 In a number of neurodegenerative and cardiovascular diseases, the production of reactive oxygen species (ROS) exceeds the antioxidant defenses and results in tissue injuries. 3,4 In this context, efforts have been directed toward the search of low-molecular-weight SODs mimics as catalytic agents for the prevention of oxidative stress disturbances. 5−8 Several Mn-based complexes have shown protective efficacy for ROS-associated pathologies 9,10 and better bioavailability than exogenous SOD enzymes. 11 Among them, Mn complexes of the salen (1,2bis(salicylidenamino)ethane) family have been tested in several animal models. 12−14 The SOD activity of mononuclear Mn-Schiff-base complexes has been extensively studied. 15 However, the mechanism of the catalyzed O 2 •− dismutation by these compounds, the influence of second-sphere effects which can be crucial for determining the reactivity of biomimetic metal complexes, 16,17 and even the oxidation states involved in the catalytic cycle remain exiguously understood. Although a Mn(II)/Mn(III) cycle during catalysis has been invoked, 18 the key features for their activity (intermediates or active species formed upon reaction with O 2 •− ) are still elusive. In this work, we try to answer two questions that remain open: do the Mn-Schiff base complexes react with O 2 •− through an outer or inner sphere mechanism? Are the redox potential of these complexes key for their reactivity? For this, we correlate the redox and spectroscopic properties of a series of Mn-X-salpn complexes ( Figure 1) with their SOD activity, aimed at unraveling some clues for their mode of action to face O 2 •− .

■ RESULTS AND DISCUSSION
Characterization of the Complexes. X-ray structures of complexes 1−4 have been reported previously. In these compounds, the metal ion is in the Mn(III) oxidation state with the ligand disposed in the equatorial plane and two capping water molecules occupying the trans-diapical positions. 19−23 The IR spectra of these complexes show the fingerprint typical of the ligands with the CN bond stretching shifted to frequencies lower than in the ligand denoting coordination to the metal ion and intense bands corresponding to the counter anions ( Figure S1). In aqueous or methanolic solution, these complexes behave as 1:1 electrolytes and can be formulated as [Mn(X-salpn)(solv) 2 ] + . Electrospray ionization (ESI)-mass spectra of the complexes in methanol show one main peak (positive mode, Figure S2) belonging to [Mn(X-salpn)] + , and the paramagnetic 1 H NMR spectra of the four compounds in D 4 -methanol exhibit a common pattern outside the diamagnetic region ( Figure S3). One broad signal at 19 ppm attributed to the central methylene of the −(CH 2 ) 3 − aliphatic chain, and one (H4/ H4′ in 2, 3, 4) or two (H4/H4′ and H5/H5′ in 1) upfield resonances belonging to the aromatic ring protons, a probe for the symmetrically arranged tetradentate Schiff-base ligand in the equatorial plane. 24,25 The −NCH− imino H appears at −95 ppm for complex 1 and −106 ppm for 4 (shown in the inset of Figure S3). Other protons adjacent to the donor groups of the Schiff base ligand (aromatic H3/H3′ and H6/ H6′ and −CH 2 −NC) are not observed in the 1 H NMR spectra (rapid relaxation or very large chemical shift). 19,24,26 Complex 5 was synthesized by adding Mn(OAc) 2 ·4H 2 O to the fully deprotonated ligand. Under these conditions, acetate binds to the Mn ion affording the neutral [Mn(5-(NO 2 )salpn)(OAc)(H 2 O)] (5). The X-ray diffraction structure of 5 (not reported previously) shows the hexacoordinated Mn(III) center adopting a pseudotetragonal geometry, with the N 2 O 2donor atoms from the tetradentate 5-(NO 2 )salpn ligand in the equatorial plane, and the apical positions occupied by one O atom from a monodentate acetate ion and one capping water molecule ( Figure 2 2 ], which in methanol behaves as a nonelectrolyte (same conductivity as for the neat solvent), indicating that the compound remains neutral in solution. The ESI-mass spectrum of a solution of 6 in DMSO shows one main peak (positive mode, Figure S2) that can be assigned to the in situ oxidized [Mn (3,5- The oxidation state of Mn in the six compounds was confirmed by perpendicular-mode electron paramagnetic

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Article resonance (EPR) spectroscopy. Complexes 1−5 are EPR-silent in frozen DMSO solutions, a fact consistent with the presence of d 4 Mn(III) ions with high zero-field splitting values. 27,28 In contrast, complex 6 exhibits a six-line EPR signal (hyperfine splitting of ∼90 G) at g = 2 and two weak resonances on the low-field side at g ≈ 3 and 5, characteristic of Mn(II) (shown below in Figure 8a) with zero-field splitting slightly weaker than the X-band microwave frequency. 29−32 Effect of the Substituents on the Reduction Potentials of Complexes 1−6. Cyclic voltammetry was used to investigate the electrochemical properties of complexes 1−6 in dimethylformamide (DMF) (Figure 3). The six complexes exhibit a quasi-reversible wave corresponding to the Mn(III)/Mn(II) redox couple, with ΔE p in the range 69− 108 mV for complexes with unsubstituted and halogensubstituted ligands, and larger ΔE p values for the nitroderivatives (Table S1). For all the six complexes, the I pa /I pc ratio is within the range 0.89−1.00. These data indicate quasireversible electrochemical processes. The phenol ring substituents span the redox potentials (E 1/2 , Table 1) of the Mn(III)/Mn(II) couple from −0.153 to 0.476 V versus saturated calomel electrode (SCE) as the electron-withdrawing ability of the substituent increases. The plot of E 1/2 versus the combined Hammett parameters of the substituents ortho and para to the phenolate (Σσ) 33 yields an excellent linear correlation ( Figure 3). This means that both substituents modulate the oxidation state of the Mn ion. This is especially evident for complex 6, where the introduction of the second NO 2 group increases the potential enough to stabilize the Mn(II) oxidation state. Besides, for complex 5 it should be expected that the axial acetate has an effect on the Mn(III)/ Mn(II) redox couple. However, the redox potential of 5 does not move away from the linear trend, suggesting that even in DMF the acetate dissociates.
Effect of the Substituents on the Charge Transfer Band in the Electronic Spectra. UV−vis spectroscopy shows that the substituent on the phenolato influences the energy of the charge transfer (CT) transitions of Mn-X-salpn complexes and provides a clear correlation between the ligand donor ability and the electron density of the metal center in these complexes. Electronic spectra of complexes 1−6 were registered in the aqueous phosphate buffer employed for the SOD tests (Figure 4a). Spectra taken immediately and 30 min (the illumination time used in the SOD tests) after preparation of solutions of 1−4 were virtually identical, meaning the complexes are stable in the buffer during the reaction time. Absorptions between 350 and 450 nm correspond to ligand to metal CT (LMCT) transitions from a pπ orbital of the phenolato oxygen to the partially filled dπ orbitals of the Mn ion. 34    6450, 1 (H)) > 26 595 cm −1 (ε = 5900, 2 (5-Cl)) > 26455 cm −1 (ε = 8300, 3 (3,5-diF)) > 25 445 cm −1 (ε = 8350, 4 (3,5-diCl)). The plot of the LMCT energy values against the combined Hammett parameter of the substituents gives a good linear correlation (Figure 4b), highlighting these ligands are predominantly π-donor: the stronger the electron-withdrawal from the O phenolato , the weaker π-donor ability of the ligand (partially filled dπ* orbitals lying at lower energy).
Effect of the Substituents on the Chemical Shift of the Aromatic H4/H4′ Protons in 1 H NMR Spectra of Complexes 1−4. The isotropically shifted NMR resonances of aromatic H4/H4′ protons of 1−4 in D 4 -methanol are compared in Figure 5. Given the low solubility of complexes 5 and 6 in methanol, these compounds were not analyzed by this technique. As stated above, the 1 H NMR spectral pattern of 1−4 indicates that in solution these complexes retain the transdiaxial geometry with the tetradentate Schiff-base ligand symmetrically arranged in the equatorial plane. 24,25,36,37 In these complexes, the phenolato protons are expected to be predominantly influenced by two contact shift pathways involving spin-delocalization onto the phenyl ring through the imino N-donor and phenolato O-donor atoms that generate upfield-shifted aromatic proton signals with large relaxation values. 24,38 The chemical shift of H4 correlates with the combined Hammett parameter of the substituents, moving downfield with the increased electron-withdrawal effect of the substituents. The H4 resonance can be used as a signature of the effect of substituent for this kind of compounds; therefore, more positive (or less negative) chemical shifts are expected for the NO 2 -derivatives.
Effect of the Substituents on SOD Activity of 1−6. The redox potential of the six complexes fall in between the potentials for the O 2 •− one electron reduction and oxidation: SCE at pH 7), respectively, so they are expected to be active for O 2 •− dismutation. The SOD activity of complexes 1−6 was evaluated in phosphate buffer of pH 7.8 using an indirect assay, which involves inhibition of the reduction of nitro blue tetrazolium (NBT) by O 2 •− . In this assay, reaction of photoreduced riboflavin with O 2 generates O 2 •− that converts the colorless NBT into purple formazan, measured at 560 nm. The SOD activity of the mimics is inversely related to the amount of formazan and the six complexes showed increasing inhibition of the reduction of NBT as their concentration raised (Figure 6a).
The IC 50 values (the concentration of the SOD mimic that diminishes by 50% the speed of reduction of NBT) were evaluated graphically and used to calculate the McCord− Fridovich second-order rate constants, k McF = k NBT [NBT]/ IC 50 (Table 1) )] + ), probably because the bending of salpn ligands leads to a weakly coordinate exchangeable axial solvent molecule which favors substrate binding. Besides, the SOD activities of these Mn-Schiff base complexes are in the same range as for Mn-amine/pyridine complexes. 15,48 The plot of log(k X /k H ) against the combined Hammett parameters of substituents (Figure 6b), where k X /k H is the ratio between k McF of X-substituted and the unsubstituted Mn complex, shows that the rate of the reaction of the Mn(III) complexes with O 2 •− decreases as the electron-withdrawal effect of the substituents increases. Because the complexes with the more electron-withdrawing substituents are more difficult to oxidize, the observed trend suggests that oxidation of the catalyst with simultaneous O 2 •− reduction should be the ratelimiting step in the reaction. 49,50 It must be noted that the diNO 2 complex (6) reacts faster than expected probably because this complex catalyzes O 2 •− dismutation through a different redox cycle (see below). On the other hand, the transeffect of bound acetate in complex 5 is expected to increase the O 2 •− reaction rate. 51 However, it follows the trend and reacts

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Article slower than complexes 1−4. Dissociation of the apical acetate ligand in the aqueous buffer medium explains this, in line with its electrolytic behavior in methanol. EPR and UV−Vis Monitoring of the Reaction of Mn-X-Salpn Complexes with KO 2 . KO 2 is rather stable toward self-dismutation in DMSO, so this solvent was chosen to follow the reaction of Mn-X-salpn complexes with superoxide. We selected complexes 1 and 5 (with the highest and lowest k McF values in this series) and 6 (in a different starting oxidation state) for the EPR monitoring of their reaction with KO 2 . As stated before, 1 is EPR-silent in perpendicular mode, whereby this technique is particularly useful to detect species in different oxidation states generated during the redox reaction. The reaction of 6 (1.6 mM) and KO 2 in DMSO was also monitored by EPR spectroscopy. The six-line signal of the starting Mn(II) complex (Figure 8a) disappeared immediately after mixing and converted into a broad signal (width = 1260 G) centered at g = 2 (Figure 8b,c) that remained several minutes and then cleared up. In this case, solid KO 2 was added to the solution of complex in DMSO, so the signal of unreacted KO 2 (g ∥ = 2.1021, g ⊥ = 2.003) 57 is observed in the spectra. The O 2 •− signal is still present when the broad signal disappears and holds during several hours. The broad unresolved signal observed in the 6 + KO 2 reaction (the signals of intermediates formed by reaction of 1, 5, and 6 with KO 2 are compared in Figure 8f) suggests that the dimerization process does not occur through a Mn(III)Mn(IV) intermediate, as in the case of 1 and 5.
To check if the formation of the dimer was related to the initial concentration of the starting complex, a more diluted sample of 1 was treated with KO 2 in DMSO and the reaction was followed spectrophotometrically. After addition of 1.5 mM KO 2 to 0.04 mM 1 in DMSO, the CT band of the starting complex (red line in Figure 9a) shifted 17 nm toward longer wave lengths (black lines in Figure 9a) and its intensity     (Figure 9b). At longer times, this band vanishes and gives rise to a band shifted toward shorter wavelengths (orange line in Figure 9a) which remains unchanged with time. This final species, which could correspond to a Mn(III) dimer, 24 is still active, as second and third additions of excess KO 2 cause the growth of the band at 395 nm and consumption of KO 2 .
Two important observations can be pointed out from these results: first, in the less concentrated solutions of 1, the Mn(IV) dimer does not form (no band at 500 nm characteristic of a dimer is observed) and, second, the complex acts as a catalyst, because it reacts with excess O 2 •− . Therefore, catalyst self-inhibition occurs at high complex concentrations by the formation of the catalytically inactive dimer, while in diluted solutions the complex behaves as a catalyst supporting several additions of excess O 2 •− , which validates concentrations employed in the SOD assays.
Proposed Mechanism for the Reaction of Mn-X-Salpn Complexes with KO 2 . A complex with the metal-centered redox couple closest to the midpoint between the oxidation and reduction of O 2 •− (0.12 V vs SCE) is expected to be a more potent SOD mimic than a complex with a farther redox potential. 15 This is essential when the reaction of O 2 •− with the complexes follows an outer sphere electron-transfer process. However, it seems not to be the case for Mn-X-salpn complexes because those complexes with redox potentials closed to 0.12 V react slower than 1 and 6. The nucleophilicity of O 2 •− and the presence of labile solvent molecules in the axial positions of complexes 1−6 favor coordination of O 2 •− followed by an inner-sphere electron-transfer mechanism, such as observed for other Mn complexes with the ligand (either cyclic or acyclic) disposed in the equatorial plane. 58−60 In such a mechanism, the variation of the electronic properties of the substituents can control the dismutation rate by modulating the strength of the Mn−O bond in the Mn−O 2 + adduct. Therefore, electron-withdrawing substituents attenuate the activity of the complex, giving rise to a weaker Mn−O 2 + bond that leads to slower electron-transfer rate. It is expected that Mn-X-salpn complexes with electron-donor substituents lead to a stronger Mn−O 2 + with higher activity. At present, we are performing density functional calculations on X-salpn-Mn + and X-salpn-Mn-O 2 + to confirm this hypothesis. On the basis of spectroscopic and kinetic results, the mechanism of Scheme 1 is proposed. At low complex concentrations, where the complexes catalyze O 2 •− dismutation, the starting complexes 1−5 may react with O 2 •− to form the Mn(IV)-peroxo adduct 61 which upon reaction with a second O 2 •− affords Mn(III)peroxo and O 2 . Proton-assisted release of HO 2 − can then take place to restore Mn(III). 62 This last step is fast in the aqueous medium of the SOD assay but much slower in DMSO, where the source of H + is the molecules of water originally present in the starting complexes. In DMSO, when the concentration of complex is ≥0.75 mM, the Mn(IV)-peroxo adduct formed in the first step can react with the Mn(III) complex to generate the Mn(III)Mn(IV) mixed valence complex (16-lines EPR spectra in Figures 7a and 8) by nucleophilic attack of the peroxo ligand on the Mn(III) center, which then evolves to the final Mn(IV) dimer (UV−vis spectrum in Figure 7d(ii)).
For 6, a lower redox cycle is proposed (Scheme 2) based on the oxidation state of Mn in the starting complex and the high redox potential expected for the Mn(III)/Mn(IV) couple. In the first step, reaction of 6 with O 2 •− should lead to formation of a Mn(III)-peroxo adduct, which reacts with a second O 2 •− radical to generate O 2 and Mn(II)-peroxo, followed by protonation to restore Mn(II) and release of HO 2 − . At higher complex concentration, the formation of a Mn(III)Mn(II) mixed valence complex is also possible through the nucleophilic attack of the Mn(III)-peroxo to the Mn(II) center of 6. 48,63 The disappearance of the broad signal in the EPR spectra taken at long times suggests that the mixed valence dimer finally yields a homovalent one, probably a Mn(III) dimer.
In conclusion, second-sphere effects of the substituents in Mn(III)-X-salpn complexes modulate the redox potentials of the metal center and the metal−ligand bond strength and guide their reactivity with O 2

•−
. Given the correlation between rates and redox potentials and the spectroscopic evidence for the formation of higher valence Mn complexes upon reaction with O 2 •− , the dismutation of O 2 •− catalyzed by 1−5 is proposed to involve Mn(III)/Mn(IV) oxidation states through an inner-sphere mechanism, where coordination of superoxide is critical. In complex 6, the two nitro groups shift the redox potential to positive values enough to catalyze O 2 •− dismutation through the Mn(II)/Mn(III) redox couple. Therefore, not only the redox potentials but also the coordination of superoxide are key for the SOD activity of these complexes.   Physical Measurements. UV−visible spectra were recorded on a Jasco V-550 spectrophotometer, with thermostated cell compartments. EPR spectra were obtained at 115 K on an Elexsys E 500 Bruker spectrometer, operating at a microwave frequency of approximately 9.5 GHz. IR spectra were recorded on a PerkinElmer Spectrum One FT-IR spectrophotometer. Solid samples were run in attenuated total reflectance mode on a diamond crystal. Metal content was determined by atomic absorption measurements on a Metrolab 250 AA spectrophotometer. ESI-mass spectra were obtained with a Thermo Scientific LCQ Fleet. The solutions for electrospray were prepared from solutions of the complexes diluted with methanol or acetonitrile to a ≈10 −5 M concentration. Complex 6 was initially dissolved in DMSO and then diluted with acetonitrile. 1 H spectra were recorded on a Bruker AC 300 NMR spectrometer at ambient probe temperature (ca. 26°C). Chemical shifts are referenced to (CH 3 ) 4 Si ( 1 H NMR) and downfield shifts are given a positive sign. Paramagnetic NMR spectra were acquired using super WEFT sequence, with acquisition time of 23 ms. The electrochemical experiments were performed with a computer-controlled Princeton Applied Research potentiostat, VERSASTAT II model, with the 270/250 Research Electrochemistry Software. Studies were carried out under Ar, in DMF solution using 0.1 M Bu 4 NPF 6 as a supporting electrolyte and ≈10 −3 M of the complexes. The working electrode was a glassy carbon disk, and the reference electrode was a calomel electrode isolated in a fritted bridged with a Pt wire as the auxiliary electrode. All potentials are referred to the SCE electrode. Under these conditions, E (ferrocene/ferrocenium) = 474 mV, in DMF.
Indirect SOD Assay. The SOD-like activity of the complexes was evaluated by measuring the inhibition of the reduction of NBT by O 2 •− spectrophotometrically. 65 50,66 EPR Measurements in DMSO. A mixture of 1 (1.25 mM) and KO 2 (1.2 mM) in DMSO was left to react and aliquots were extracted at different times and frozen in N 2 (l) for EPR measurements. For 5 + KO 2 , the concentrations were 0.75 and 1.1 mM, respectively.
A mixture of 0.16 mL of a DMSO solution of 6 (1.6 mM) and 0.1 mL of saturated solution of KO 2 (with excess solid KO 2 ) was left to react and aliquots were extracted at different times and frozen in N 2 (l) for EPR measurements.
Stock KO 2 solution in anhydrous DMSO was prepared by mixing 9.3 mg of KO 2 in DMSO (5 mL) and sonicated for 15 min, followed by centrifugation at 6000 rpm during 25 min. The concentration of KO 2 in the supernatant was estimated by using its extinction coefficient 2686 M −1 cm −1 in deoxygenated DMSO solution 67 and confirmed by the horseradish peroxidase assay.
Crystal Data Collection and Refinement. Single-crystal data were collected on an Oxford Diffraction Gemini E CCD diffractometer equipped with a sealed tube with Mo Kα radiation (λ = 0.71073 Å). Crystal structure data were corrected for absorption with CrysAlisPro, Agilent Technologies, version 1.171.34.49, applying an empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. The structure was solved by Patterson methods with SHELXS-97 68 and refined by fullmatrix least-squares on F2 with SHELXL-97. 69 All nonhydrogen atoms were refined anisotropically and hydrogen atoms were added geometrically and refined as riding atoms with a uniform value of U iso . Crystal data collection and refinement parameters for compound 5 are summarized in Table S2.