Reactions of a Chromium(III)-Superoxo Complex and Nitric Oxide That Lead to the Formation of Chromium(IV)-Oxo and Chromium(III)-Nitrito Complexes

Abstract

The reaction of an end-on Cr(III)-superoxo complex bearing a 14-membered tetraazamacrocyclic TMC ligand, [Cr^III(14-TMC)(O₂)(Cl)]⁺, with nitric oxide (NO) resulted in the generation of a stable Cr(IV)-oxo species, [Cr^IV(14-TMC)(O)(Cl)]⁺, via the formation of a Cr(III)-peroxynitrite intermediate and homolytic O-O bond cleavage of the peroxynitrite ligand. Evidence for the latter comes from EPR spectroscopy, computational chemistry, and the observation of phenol nitration chemistry. The Cr(IV)-oxo complex does not react with nitrogen dioxide (NO₂), but reacts with NO to afford a Cr(III)-nitrito complex, [Cr^III(14-TMC)(NO₂)(Cl)]⁺. The Cr(IV)-oxo and Cr(III)-nitrito complexes were also characterized spectroscopically and/or structurally.

Nitric oxide (NO) is an ubiquitous signal transduction molecule which is found in a wide variety of physiological processes such as a cellular signaling leading to smooth muscle vasodilation, platelet disaggregation, and neurotransmission and in the immune response to bacterial infection.¹ However, NO itself is a toxin because of the reactivity derived from its radical character and ability to form more reactive and deleterious oxidation products, such as NO₂ and peroxynitrite (PN, oxoperoxonitrate (1–), ⁻OON=O), in the presence of transition metals.² In this context, various life forms have constructed detoxification systems; widely occurring nitric oxide dioxygenases (NODs) are enzymes which catalyze the conversion of toxic NO to biologically atoxic nitrate (NO₃⁻) with NAD(P)H (i.e., 2NO + 2O₂ + NAD(P)H → 2NO₃⁻ + NAD(P)⁺ + H⁺).³ In NOD reactions, including those observed with hemoglobins (Hbs) and myoglobins (Mbs), an Fe(III)-superoxo species (Fe(III)(O₂^•−)), which is generated in the reaction of Fe(II) + O₂, reacts with NO to yield nitrate via formation of an Fe(III)-peroxynitrite intermediate.^4,5 Here, a ferryl species (Fe(IV)(O)) is proposed to be formed via homolytic O-O bond cleavage of the Fe(III)-peroxynitrite species; Groves and co-workers only very recently provided time-resolved spectrophotometric evidence for the formation of such a ferryl species in the reaction of Mb with PN.⁶ However, a heme-PN moiety has not been detected due to its expected short lifetime and rapid recombination with NO₂ in a [Fe^IV=O •NO₂] cage to give a nitrate product. Thus, the study of metal-superoxo intermediates with NO is of great interest with respect to the investigation of reaction pathways or undetected intermediate reactivity, such as in the possible escape of a bound PN or NO₂ molecule.⁷

Recently, TMC (N-tetramethylated cyclam)⁸ chelated redoxactive first row transition metal complexes and their peroxo (O₂²⁻) and superoxo (O₂^•−) derivatives have been systematically synthesized and investigated in various oxidation reactions.^9-12 Especially, in the case of Ni and Cr complexes with the 14-TMC¹³ and 12-TMC¹³ ligands, the TMC ring size was found to alter the structure of the resulting [M(n-TMC)(O₂)]ⁿ⁺ complexes; the larger ring size ligand (e.g., 14-TMC) forms metal-superoxo complexes (e.g., Ni(II)-superoxo^10b and Cr(III)-superoxo¹¹), whereas the smaller ring size 12-TMC chelate forms metal-peroxo complexes (e.g., Ni(III)-peroxo^10b and Cr(IV)-peroxo¹²). In addition, we recently reported the NOD reactivity of the Cr(IV)-peroxo complex; this species reacted with NO to give a Cr(III)-nitrate product, [Cr^III(12-TMC)(NO₃)(Cl)]⁺, however, without detection of any intermediates such as Cr-PN or Cr-oxo species.¹²

We herein report the reactions of the end-on Cr(III)-superoxo complex bearing the 14-TMC ligand, [Cr^III(14-TMC)(O₂)(Cl)]⁺ (1), with NO. Complex 1 with one equiv. of NO generates a stable Cr(IV)-oxo species, [Cr^IV(14-TMC)(O)(Cl)]⁺ (3), via the formation of a presumed Cr(III)-peroxynitrite intermediate, [Cr^III(14-TMC)(OON=O)(Cl)]⁺ (2) (Scheme 1, reactions a and b), unlike the NO reaction of a Cr(IV)-peroxo complex bearing the 12-TMC.¹² To the best of our knowledge, this study presents the first strong evidence for the formation of a high-valent metal-oxo complex in the reaction of a metal-superoxo species and NO. Further, 3 does not lead to a nitrate containing product with NO₂ addition, but it does react with NO to give a Cr(III)-nitrito complex, [Cr^III(14-TMC)(NO₂)(Cl)]⁺ (4) (Scheme 1, reaction c). Thus, we were able to follow all the reaction pathways of metal-oxygen intermediates with NO, employing isolated or spectroscopically well-characterized Cr complexes.

Scheme 1.

The starting chromium complex, [Cr^II(14-TMC)(Cl)]Cl, was synthesized according to literature methods, and the Cr(III)-superoxo complex (1) (2 mM) was prepared by bubbling O₂ for 30 s in an CH₃CN solution containing the starting complex at −40 °C, giving a dark red color.¹¹ After purging the reaction solution with Ar for 30 min at −40 °C, one equiv. of NO¹⁴ was added and stirred for 20 min (see Supporting Information (SI), Figure S1 for a schematic diagram showing the NO purification setup), whereupon the color of the solution changed from red to yellowish green (see Figure 1 for the UV-vis spectral changes). In this experiment, the spectrum of 1 changed from blue solid line to the black dashed line immediately upon addition of NO, followed by the conversion of this intermediate (2) to the Cr(IV)-oxo species (3, red line) with λ_max at 603 nm (80 M⁻¹ cm⁻¹) over 15 min (Figure 1). This final absorption spectrum is consistent with our previous report on the Cr(IV)-oxo complex with this 14-TMC ligand,¹¹ and the conversion of 1 to 3 turned out to be quantitative. A resonance Raman (rRaman) spectrum of 3 was collected using 407-nm excitation in CH₃CN at −20 °C (SI, Figure S2). The peak observed at 874 cm⁻¹ is comparable to the Cr–O frequency (873 cm⁻¹) of the previously reported [Cr^IV(14-TMC)(O)(Cl)]⁺ complex.¹¹ The electrospray ionization mass spectrum (ESI-MS) of 3 also supports the generation of Cr(IV)-oxo species; the observed peak cluster centered at m/z 359.3 can be assigned to a Cr(IV)-oxo complex formulated as [Cr^IV(14-TMC)(O)(Cl)]⁺ (calcd. m/z of 359.2) (SI, Figure S3). An electron paramagnetic resonance (EPR) spectrum obtained for 3 (1 mM) in CH₃CN at 5 K was silent, consistent with the expected oxidation state of Cr^IV in 3. Based on the spectroscopic characterization of 3, we were able to conclude unambiguously that a highvalent Cr(IV)-oxo species was formed in the reaction of this Cr(III)-superoxo complex and nitric oxide.

Figure 1.

UV-vis spectral changes of 1 (2 mM) upon addition of 1 equiv. NO in CH₃CN at −40 °C under Ar. The initial blue solid line spectrum (1) changed immediately to the black dashed line spectrum (2) upon addition of NO, followed by the conversion to the red solid line spectrum (3) over 15 min. Inset; time-dependent EPR spectra of Cr(III)-PN (2) in CH₃CN at 5 K; 1 and NO are mixed in CH₃CN at −40 °C under Ar and EPR spectra were obtained by freezing such solutions as a function of time (0, 30, 60, 100, 150, and 300 sec).

A proposed mechanism for the generation of Cr(IV)-oxo species (3) in the reaction of Cr(III)-superoxo (1) and NO is then depicted in Scheme 1. According to widely accepted discussions on the reaction mechanism of Fe(III)-superoxo species with NO,^2-6 the first step of the reaction must be a radical coupling reaction between 1 and NO to generate a Cr(III)-peroxynitrite intermediate (2, Cr^III-PN), [Cr^III(14-TMC)(OON=O)(Cl)]⁺ (Scheme 1, reaction a). This species gives rise to 3 and NO₂ via O-O bond homolysis of the PN ligand (Scheme 1, reaction b). The Cr(IV)-oxo species generated does not further react with the NO₂ produced simultaneously, unlike that which occurs for NOD reactions,^3-6 and it maintains its metal-oxo structure under the reaction conditions.

We then attempted to characterize the presumed Cr(III)-peroxynitrite intermediate (2, Cr^III-PN) and elucidate the mechanism of the conversion of 2 to the Cr(IV)-oxo species (3) (Scheme 1, reaction b). First, the UV-vis spectrum of the black dashed line in Figure 1 likely represents that of 2. Notably, the dashed line spectrum and the following changes shown in Figure 1 were not observed when the reaction was carried out at a higher temperature (e.g., 0 °C). For this latter experiment, the spectrum changed instantly from the blue solid line (1) to the red line (3) without showing any intermediate spectra, implying that the stability of 2 depends on the reaction temperature and 2 is detectable only at a low temperature. We also found via EPR measurements that the intermediate species (i.e., the black dashed line in Figure 1) exhibited a signal assignable to a d³ Cr(III) ion (S = 3/2) at 5 K (SI, Figure S4a),¹⁵ while complexes 1 and 3 were EPR silent. Moreover, time-dependent EPR measurements reveal the clear conversion of 2 to 3; EPR spectra in CH₃CN at 5 K obtained by freezing solutions of the 1 + NO reaction mixture as a function of time (0 to 300 sec) indicated that the signal intensity decreased as time passed, leading from EPR active 2 to EPR silent 3 (Figure 1, inset). Thus, the Cr(III) EPR signal detected for 2 supports the generation of the Cr(III)-PN species, which decomposes to the EPR silent species 3.¹⁶ As reported previously, the lifetimes of metal-peroxynitrite species are very short for near RT generated species.¹⁷ A striking recent success is a Co(III)-peroxynitrite porphyrinate complex, yet still generated under cryogenic conditions, but thoroughly characterized by using IR-spectroscopy and isotope labeling experiments along with DFT calculations.¹⁸

Density functional theory (DFT) calculations were also performed in order to seek further support for our suppositions concerning the intermediacy of Cr(III)-PN complex 2. Complex 1 is an S = 1 state species,^9a,11b but by adding the NO spin (up or down), the whole system can reside in two energetically degenerate states, S = 1/2 or S = 3/2. The calculations reveal that the S = 1/2 state features a concerted high-energy barrier for the current reaction (Figure 2) and is therefore not discussed further.¹⁹ The S = 3/2 state reaction pathway is shown to be able to proceed in two low-energy steps. The first step of the reaction occurs over a low barrier of 1.7 kcal/mol, such that a very fast reaction of 1 with NO would occur, and alternative reactions are unlikely. Moreover, the product is a stable O-bound peroxynitrite species 2 at −10.8 kcal/mol (SI, Figure S5). The next step of the reaction occurs over a barrier of 20.1 kcal/mol to form the Cr(IV)-oxo complex 3. This barrier is somewhat high, but is likely a well overestimated value.²⁰ All in all, the interpretation of the calculations strongly support that the formation of the S = 3/2 species 2 is likely, and its transformation to 3 is quite possible.

Figure 2.

DFT-calculated pathways for the reaction of [Cr^III(14-TMC)(O₂)(Cl)]⁺ (1) with NO. The low-energy two-step pathway giving PN complex 2 and then Cr^IV=O species 3 occurs on the S = 3/2 surface (red, ■), while the S = 1/2 surface (green, ●) exhibits a concerted pathway that does not feature a (stable) peroxynitrite intermediate (2).

We sought more support for our mechanistic proposal, including the formation of 3 from 2 with the liberation of NO₂. In this regard, we carried out trapping experiments using 2,4-di-tert-butylphenol (DTBP) to obtain evidence that NO₂ is produced concomitantly with the Cr(IV)-oxo formation (Scheme 1, reaction b).²² After the generation of 3 as discussed above, 1 equiv. of DTBP was added to the resulting solution and stirred for 20 min under Ar, while also monitoring the reaction’s UV-vis spectral changes (see SI, Figure S6). ESI-MS analysis of the resulting solution revealed the presence of a Cr(III)-hydroxo species at m/z 360.2 ([Cr^III(14-TMC)(OH)(Cl)]⁺, calcd. m/z of 360.3) (SI, Figure S6). Further, product analysis was performed using GC and ¹H NMR spectroscopic measurements; nitrated DTBP (2,4-di-t-butyl-6-nitrophenol, nitro-DTBP) and oxidatively dimerized DTBP (2,2’-dihydroxy-3,3’,5,5’-tetra-t-butylbiphenol, DTBP-dimer) products were found in 46% and 12% yields, respectively (see SI, Experimental Section). The results can be readily explained as follows: First, H-atom abstraction reaction of DTBP by 3 gives a phenoxyl radical and a Cr(III)-hydroxo complex (Scheme 2, reaction I). The phenoxyl radical traps gaseous NO₂ to give nitro-DTBP (Scheme 2, reaction II) or dimerizes to give DTBP-dimer (Scheme 2, reaction III). Thus, the results that we successfully trapped and recovered the NO₂ in good yield also strongly support the proposed reaction mechanism involving the formation of a Cr(IV)-oxo complex and NO₂ via the homolytic O-O bond cleavage of a Cr^III-PN intermediate (Scheme 1, reaction b).

Scheme 2.

In heme and nonheme systems, the reaction of a ferryl species and NO has been of great interest in terms, for example, in the inhibition of cytochrome bd oxidase reactivity;²³ here, a ferryl heme species reacts with NO to generate a ferric species and nitrite (Fe(III) + NO₂⁻),^23,24 while a ferryl nonheme complex ([Fe^IV(14-TMC)(O)(OAc)]⁺) with added NO produces a ferrous species and nitrite (Fe(II) + NO₂⁻).²⁵ The reactions of other metal-oxo complexes and NO have also been discussed.²⁶ Since we did not observe nitrate (NO₃⁻) formation via a coupling between the Cr(IV)-oxo complex (3) and NO₂ (vide supra) and the formation of a Cr(III)-nitrito complex, [Cr^III(14-TMC)(NO₂)(Cl)]⁺ (4), was observed when an excess amount of NO was used in the reaction of a Cr(III)-superoxo complex (1), we decided to check if the Cr(IV)-oxo complex 3 could react with NO, not with NO₂, to form 4 (Scheme 1, reaction c). Indeed, this is the case. After generating 3 as described above, the solution was degassed by Ar-bubbling to remove NO₂ (vide supra), and then excess NO was added over the top of the solution at −40 °C. The UV-vis spectral changes for 3 upon addition of NO and the ESI-MS of the resulting solution are depicted in Figure 3; the characteristic absorption band of 3 at 603 nm disappeared gradually over 5 min, and a molecular ion peak corresponding to 4 appeared at m/z 388.9. When 1 was prepared with isotopically labeled ¹⁸O₂ and used in the subsequent reaction with NO, a peak corresponding to [Cr^III(14-TMC)(N¹⁸O¹⁶O)(Cl)]⁺ (calcd. m/z of 391.2) was found at m/z 390.9 (Figure 3, inset). The two mass unit shift observed upon substitution of ¹⁶O with ¹⁸O indicates that 3 has one ¹⁸O oxygen (out of two) in it and its origin is the Cr(III)-superoxo complex (1) via Cr(IV)=O species (3) (Scheme 1, reaction c). Additionally, an EPR spectrum of 4 in CH₃CN at 5 K showed a signal assigned to a d³ Cr(III) ion (S = 3/2)¹⁵ (SI, Figure S3b). Based on the results presented above, we conclude that one electron reduction in Cr occurs through the reaction between 3 and NO, resulting in the formation of [Cr^III(14-TMC)(NO₂)(Cl)]⁺ (4; SI, Figure S6).

Figure 3.

UV-vis spectral changes of 3 (black line) upon addition of NO (10 cm³) into the headspace above a solution of 3 in CH₃CN at −40 °C under Ar. Inset; ESI-MS spectrum of [Cr^III(14-TMC)(NO₂)(Cl)]⁺ (4) (calcd. m/z of 389.1) and isotope distribution patterns for 4-¹⁶O (red) and 4-¹⁸O (blue). The peaks at 359.1 and 378.1 with asterisk are assigned to [Cr^IV(14-TMC)(O)(Cl)]⁺ (calcd. m/z of 359.2) and [Cr^III(14-TMC)(Cl₂)]⁺ (calcd. m/z of 378.1), respectively.

In summary, we have demonstrated the generation of a stable Cr(IV)-oxo species (3) in the reaction of a Cr(III)-superoxo complex (1) and nitric oxide. This is the first clear observation of metaloxo species generation in the reaction of a metal-superoxo complex and NO via metal-peroxynitrite formation and its subsequent homolytic O-O bond cleavage. This opens the way to utilize such an approach to generate biochemical or synthetic higher-valent metaloxo species. Interestingly, the Cr(IV)-oxo species didn’t show any reactivity toward NO₂, unlike NOD reactions, but it readily reacted with NO to form a Cr(III)-nitrito complex (4). In addition, we have shown that the ring size of the TMC ligand has a significant effect not only on the determination of metal-O₂ structures but also on the reactivity of the resulting metal-oxo complexes (e.g., Cr(IV)-oxo species) towards NO and NO₂.