Dioxygen Activation and O–O Bond Formation Reactions by Manganese Corroles

Abstract

Activation of dioxygen (O₂) in enzymatic and biomimetic reactions has been intensively investigated over the past several decades. More recently, O–O bond formation, which is the reverse of the O₂-activation reaction, has been the focus of current research. Herein, we report the O₂-activation and O–O bond formation reactions by manganese corrole complexes. In the O₂-activation reaction, Mn(V)-oxo and Mn(IV)-peroxo intermediates were formed when Mn(III) corroles were exposed to O₂ in the presence of base (e.g., OH⁻) and hydrogen atom (H atom) donor (e.g., THF or cyclic olefins); the O₂-activation reaction did not occur in the absence of base and H atom donor. Moreover, formation of the Mn(V)-oxo and Mn(IV)-peroxo species was dependent on the amounts of base present in the reaction solution. The role of the base was proposed to lower the oxidation potential of the Mn(III) corroles, thereby facilitating the binding of O₂ and forming a Mn(IV)-superoxo species. The putative Mn(IV)-superoxo species was then converted to the corresponding Mn(IV)-hydroperoxo species by abstracting a H atom from H atom donor, followed by the O–O bond cleavage of the putative Mn(IV)-hydroperoxo species to form a Mn(V)-oxo species. We have also shown that addition of hydroxide ion to the Mn(V)-oxo species afforded the Mn(IV)-peroxo species via O–O bond formation and the resulting Mn(IV)-peroxo species reverted to the Mn(V)-oxo species upon addition of proton, indicating that the O–O bond formation and cleavage reactions between the Mn(V)-oxo and Mn(IV)-peroxo complexes are reversible. The present study reports the first example of using the same manganese complex in both O₂-activation and O–O bond formation reactions.

Graphical Abstract

1. INTRODUCTION

Dioxygen (O₂) is one of the most important molecules for sustaining aerobic life. In biological reactions, metalloenzymes bind and activate O₂, and metal–oxygen intermediates, such as metal-superoxo, -peroxo, -hydroperoxo, and -oxo species, are generated as reactive species in the catalytic cycle of O₂-activation.¹ One notable example is the O₂-activation by iron-containing heme and nonheme enzymes, in which iron–oxygen intermediates have been trapped and characterized using various spectroscopic techniques.^2,3 In biomimetic studies, a large number of iron–oxygen intermediates bearing porphyrin and nonporphyrin ligands have been successfully synthesized as the model compounds of the heme and nonheme iron enzymes, and their chemical properties as well as the mechanisms of the formation of the iron–oxygen intermediates have been elucidated using the model compounds.^4,5 In most of the model studies, artificial oxidants, such as iodosylbenzene (PhIO), peracids, NaOCl, H₂O₂, and alkyl hydroperoxides, have been used as surrogates for O₂ for the synthesis of the iron–oxygen intermediates.^4,5 However, enzymes utilize O₂, not the artificial oxidants, in most of their reactions and O₂ is an ideal, environmentally benign oxidant in oxidation reactions. Therefore, tremendous efforts have been exerted to use O₂ as the primary oxidant in biomimetic oxidation reactions, and the formation of iron–oxygen complexes by activating O₂ in the absence or presence of electron and proton donors have been reported recently.^6,7

Similar to the iron systems, high-valent manganese-oxo intermediates have been considered as the reactive species in the oxidation of organic substrates as well as in the water oxidation reaction by the oxygen-evolving complex (OEC) in Photosystem II (PS II).⁸ Therefore, a number of high-valent Mn(IV or V)-oxo complexes have been synthesized in the reactions of heme and nonheme Mn models using artificial oxidants (e.g., PhIO, peracids, and NaOCl).⁷ In addition to the Mn-oxo species, other Mn-oxygen intermediates, such as Mn-peroxo, -hydroperoxo, and -alkylperoxo species, have been synthesized using H₂O₂ and alkyl hydroperoxides as the oxidants.⁹ However, in most of the reactions, artificial oxidants were used for the synthesis of the Mn-oxygen intermediates, and there are only a couple of examples using O₂ as the oxidant for the synthesis of Mn(V)-oxo complexes.¹⁰ Moreover, to the best of our knowledge, the formation of other Mn-oxygen intermediates (e.g., Mn-peroxo species) has never been observed in the O₂-activation reactions.

The reverse of the O₂-activation reaction is the O–O bond formation reaction, in which high-valent metal-oxo species have been proposed as the active oxidants responsible for the O–O bond formation (e.g., Mn(V)-oxo species in the water oxidation by OEC).⁸ Although the water oxidation reaction has been the topic of current research and intense effort has been devoted to the development of highly efficient catalytic systems,^8,11 the nature of reactive intermediates and the mechanism(s) of the O–O bond formation still remain elusive. Only a couple of examples showing the O–O bond formation between high-valent Mn(V)-oxo species and hydroxide ion have been reported in biomimetic studies.¹² Thus, compared to the O–O bond activation chemistry, the O–O bond formation mechanism is less clearly understood. Taken together, it is indeed a big challenge to achieve both of O–O bond activation and O–O bond formation by employing a single biomimetic complex.

Herein, we report the O₂-activation and O–O bond formation reactions by manganese corrole complexes (see Scheme 1). In the O₂-activation reaction, the formation of Mn(V)-oxo and Mn(IV)-peroxo complexes was observed when Mn(III) corrole complexes were exposed to O₂ in the presence of base and hydrogen atom (H atom) donors. The Mn(V)-oxo and Mn(IV)-peroxo intermediates were characterized spectroscopically by synthesizing authentic compounds. In the O–O bond formation reaction, addition of hydroxide ion to the Mn(V)-oxo complexes yielded the Mn(IV)-peroxo species, and the reversible O–O bond formation and cleavage reactions between the Mn(V)-oxo and Mn(IV)-peroxo complexes were demonstrated. Mechanistic aspects for the O₂-activation and O–O bond formation reactions have also been discussed in the present study.

Scheme 1.

(A) Manganese–Oxygen Intermediates Formed in the O₂-Activation and O–O Bond Formation Reactions and (B) Manganese Corroles Used in This Study

2. RESULTS AND DISCUSSION

2.1. Formation of Mn(V)-Oxo and Mn(IV)-Peroxo in O₂-Activation Reactions

2.1.1. Generation of Mn(V)-Oxo by Activating O₂

The manganese(III) corrole complexes used in this study were [(TPFC)Mn^III] (TPFC = 5,10,15-tris-(pentafluorophenyl)corrolato trianion) and [(TPFcCN)Mn^III] (TPFcCN = 5,15-bis(pentafluorophenyl)-10-benzonitrilecorro-lato trianion) (see Scheme 1B for structures), which were stable in O₂ atmosphere at room temperature in THF. Interestingly, when [(TPFC)Mn^III] was exposed to air in the presence of 2 equiv of tetramethylammonium hydroxide (TMAH) in THF at 25 °C,¹³ the color of the reaction solution changed from green to pink and a new intermediate 1 with absorption bands at 347, 407, and 520 nm was formed within 2 min (Figure 1a); the electronic absorption spectrum of 1 was similar to those of [(Corrole)Mn^V(O)] complexes reported previously.¹⁴ Intermediate 1 was also synthesized as an authentic compound by reacting [(TPFC)Mn^III] with 1-(tert-butylsulfonyl)-2-iodosyl-benzene (^sPhIO) in CH₃CN at 0 °C (Scheme 2, pathway h) (Supporting Information (SI), Figure S2).¹⁴ Intermediate 1 was stable (t_1/2 ~ 3 h at 25 °C) and was characterized using various spectroscopic methods. The cold-spray ionization mass (CSI-MS) spectrum exhibited a prominent ion peak at a mass-to-charge ratio (m/z) of 864.0 (SI, Figure S3a), which corresponds to the mass and isotope distribution pattern of [(TPFC)Mn-(O)] (calcd m/z = 864.0). When the reaction was carried out with ¹⁸O₂ gas, the CSI-MS exhibited a peak at m/z 866.0 (SI, Figure S3b), which corresponds to [(TPFC)Mn(¹⁸O)] (calcd m/z = 866.0). The observation of the two-mass unit shift in the ¹⁸O-labeled experiment suggests that 1 contains one oxygen atom and the oxygen atom in 1 derives from O₂. The X-band EPR and ¹H NMR spectra of 1 indicate that 1 is diamagnetic with a low-spin (S = 0) state (SI, Figures S4 and S5), as reported in other Mn(V)-oxo complexes.^10,12,14,15 The resonance Raman (rRaman) spectrum of 1, measured at –40 °C upon 405 nm excitation, displayed one isotopically sensitive band at 958 cm⁻¹, which shifted to 918 cm⁻¹ upon ¹⁸O-substitution (Figure 1b).^12b The observed isotopic shift of –40 cm⁻¹ with ¹⁸O-substitution was in good agreement with the calculated value for a diatomic Mn–O oscillator (–42 cm⁻¹). The calculated force constant for the 958 cm⁻¹ mode by a simple Hook’s law was 6.7 mdyne/Å, suggesting that Mn–O moiety possesses triple Mn–O bond character as reported in other Mn(V)-oxo complexes.^{10c,12b,14c,15a,b}

Figure 1.

(a) UV–vis spectral changes observed in the O₂-activation by [(TPFC)Mn^III] (0.030 mM, red line) in the presence of TMAH (0.060 mM, 2 equiv) to form 1 (blue line) in THF at 25 °C. Inset shows the time trace monitored at 347 nm due to 1. (b) Resonance Raman spectra of 1-¹⁶O (blue line) and 1-¹⁸O (red line) obtained upon excitation at 405 nm. Black line is the difference spectrum of 1-¹⁶O and 1-¹⁸O.

Scheme 2.

Proposed Mechanisms for the Generation of Mn(V)-Oxo and Mn(IV)-Peroxo Species in O₂-Activation and O–O Bond Formation Reactions

Similarly, [(TPFcCN)Mn^V(O)] was formed when [(TPFcCN)Mn^III] was exposed to air in the presence of 2 equiv of TMAH in THF at 25 °C (see the structure of [(TPFcCN)Mn^III] in Scheme 1B) (SI, Figure S6a). [(TPFcCN)Mn^V(O)] was also synthesized by reacting [(TPFcCN)Mn^III] with ^sPhIO in CH₃CN at 0 °C (SI, Figure S6b). The [(TPFcCN)Mn^V(O)] complex was characterized with other spectroscopic methods, such as EPR (data not shown). Taken together, Mn(V)-oxo corrole complexes were formed in the activation of O₂ by Mn(III) corroles in the presence of base in THF at 25 °C.

2.1.2. Generation of Mn(IV)-Peroxo by Activating O₂

When a THF solution containing [(TPFC)Mn^III] (0.030 mM) and large excess TMAH (0.60 mM) was exposed to air at 25 °C, the formation of a new intermediate 2 with absorption bands at 435 and 574 nm in an electronic absorption spectrum was observed within 10 s (Figure 2a, blue line); the color of the reaction solution changed from green to yellowish brown. The X-band EPR spectrum of 2 exhibited a broad signal at g ≈ 5.1, which was assigned to a high-spin S = 3/2 Mn(IV) species (SI, Figure S7, blue line).¹⁶ Intermediate 2 was also synthesized by reacting [(TPFC)Mn^III] and H₂O₂ in the presence of TMAH (Scheme 2, pathway i) (SI, Figure S2b).¹²

Figure 2.

(a) UV–vis spectral changes observed in the O₂-activation by [(TPFC)Mn^III] (0.030 mM, red line) in the presence of TMAH (0.60 mM, 20 equiv) to form 2 (blue line) in THF at 25 °C. (b) UV–vis spectral changes showing the conversion of 1 (0.030 mM, red line) to 2 (blue line) upon addition of TMAH (0.60 mM, 20 equiv) and the regeneration of 1 (pink line) upon addition of HClO₄ (0.60 mM, 20 equiv) to the resulting solution of 2.

A comparison of the Mn K-edge XAS data for [(TPFC)Mn^III] and 2 is shown in Figure 3a. The data show that on going from the starting Mn^III species to 2, the rising-edge inflection point shifts by 2.2 eV to higher energy (from 6550.0 to 6552.2 eV). Because the rising-edge energy position shifts to higher energies with an increase in the effective nuclear charge at the absorbing Mn center,^16c,17 the data indicate a clear increase in oxidation state at the Mn site and show that 2 is a Mn(IV) species. The Mn K-pre-edge spectrum, which arises due to Mn 1s → 3d electric dipole forbidden, quadrupole allowed transitions, is strongly affected by changes in the ligand-field strength and bonding at the Mn center.^18,19 On going from [(TPFC)Mn^III] to 2, the pre-edge shifts to higher energy by 0.6 eV (6540.5 to 6541.1 eV). In addition, the pre-edge intensity pattern changes from a single sharp feature in [(TPFC)Mn^III] to a broad, weaker feature in 2. Together, these data indicate an increase in ligand-field and a more symmetrical structure in 2 relative to [Mn^III(TPFC)]. This qualitative structural assessment from the Mn K-pre-edge data was augmented with Mn K-edge EXAFS data for [(TPFC)Mn^III] and 2. Figure 3b shows a comparison of the nonphase shift corrected Fourier transform data for the two species (see Figure 3b, inset for the EXAFS comparison). The data reveal a significant increase in the first shell intensity indicating an increase in coordination at the Mn. FEFF best fits to the data for [(TPFC)Mn^III] are consistent with a first shell with 4 Mn–N at 1.91 Å,²⁰ while for 2, a split first shell is required with 2 Mn–O contributions at 1.83 Å and 4 Mn–N at 1.96 Å. For both [(TPFC)Mn^III] and 2, the second and third shells were fit with single and multiple scattering contributions from the TPFC ligand (SI, Figure S8 and Table S1). These data are in good agreement with the Mn K-pre-edge intensities and together the Mn K-edge XAS and EXAFS data are consistent with a side-on bound peroxo Mn(IV) description for 2.

Figure 3.

(a) Normalized Mn K-edge XAS data for [(TPFC)Mn^III] (black line) and 2 (red line). Inset shows the expanded pre-edge region. (b) Non-phase-shift corrected Fourier Transform data for [(TPFC)-Mn^III] (black line) and 2 (red line). Inset shows the Mn K-edge EXAFS comparison.

Similarly, we observed the formation of [(TPFcCN)-Mn^IV(O₂)]⁻ when [(TPFcCN)Mn^III] was exposed to air in the presence of 20 equiv or TMAH in THF at 25 °C (SI, Figure S9a). The [(TPFcCN)Mn^IV(O₂)]⁻ complex was also synthesized as an authentic compound by reacting [(TPFcCN)Mn^III] with H₂O₂ in the presence of TMAH in CH₃CN at 0 °C (SI, Figure S9b). On the basis of the spectroscopic data presented in this study and the previous reports,¹² we conclude that Mn(IV)- peroxo complexes were formed by activating O₂ in the presence of a large excess amount of base in THF at 25 °C.

2.1.3. Reversible O–O Bond Formation and Cleavage between Mn(V)-Oxo and Mn(IV)-Peroxo Complexes

We have shown above that Mn(V)-oxo and Mn(IV)-peroxo complexes can be formed in the O₂-activation reaction, depending on the amounts of TMAH present in the reaction solution; Mn(V)-oxo and Mn(IV)-peroxo species were formed in the presence of 2 and 20 equiv of TMAH, respectively. We propose the following mechanisms for the formation of different Mn-oxygen intermediates depending on the amounts of TMAH: In the reaction using an excess amount of TMAH (i.e., 20 equiv), 1, which was generated in the O₂-activation reaction by [(TPFC)-Mn^III] initially, reacted further with OH⁻ at a fast rate, thereby affording the formation of 2 (Scheme 2, pathway e). Indeed, the fast conversion of 1 to 2 was confirmed by carrying out a control reaction, in which addition of 20 equiv of TMAH to the solution of 1, which was generated either in the O₂-activation reaction (Figure 2) or in the reaction of [(TPFC)Mn^III] and ^sPhIO (SI, Figure S10), resulted in the immediate conversion of 1 to 2. Thus, 2 can be generated via the O–O bond formation process between Mn(V)-oxo and OH⁻, as reported previously.¹² An alternative mechanism proposing the formation of 2 in the presence of an excess amount of OH⁻ is the fast deprotonation of the putative Mn(IV)-OOH species generated in the catalytic cycle (Scheme 2, pathway f). Thus, there are two competing pathways for the reaction of the Mn(IV)-OOH species, such as the O–O bond cleavage of Mn(IV)-OOH to form 1 (Scheme 2, pathway d), followed by the O–O bond formation between 1 and OH⁻ (Scheme 2, pathway e) or the deprotonation of Mn(IV)-OOH by OH⁻ to form 2 (Scheme 2, pathway f). Because the rates of the O–O bond cleavage and deprotonation steps of the Mn(IV)-OOH species cannot be determined without capturing the Mn(IV)-OOH species, we cannot distinguish the formation mechanism of 2 from the putative Mn(IV)-OOH species at this moment (e.g., Scheme 2, pathways d versus f).

The reverse reaction of the O–O bond formation between Mn(V)-oxo and OH⁻ (Scheme 2, pathway e) is the O–O bond cleavage reaction upon addition of H⁺ to the Mn(IV)-peroxo species (Scheme 2, pathway g, followed by pathway d). Indeed, we observed the immediate formation of 1 upon addition of H⁺ to 2 (Figure 2b), as reported previously.^12,21 For the latter reaction, we propose that addition of H⁺ to 2 forms the putative Mn(IV)-OOH species (Scheme 2, pathway g), followed by the O–O bond cleavage of the Mn(IV)-OOH species to form 1 (Scheme 2, pathway d).¹²

2.2. Mechanistic Investigations

2.2.1. Role of Base in O₂-Activation Reaction

The O₂-activation by Mn(III) corroles occurs only in the presence of base (e.g., TMAH), indicating that base is an essential component in the O₂-activation reaction. We therefore investigated the role of base in the O₂-activation reaction by [(TPFC)Mn^III]. Because it is difficult to control the fast reaction of [(TPFC)Mn^III] with O₂ in THF, mechanistic studies were performed in CH₃CN; [(TPFC)Mn^III] does not react with O₂ in CH₃CN even in the presence of base when there is no hydrogen atom (H atom) donor in reaction solution (e.g., THF).

First, addition of TMAH to [(TPFC)Mn^III] in CH₃CN at 25 °C afforded the formation of a new species 3 with an absorption band at 471 nm (SI, Figure S12); this species was stable under an air atmosphere. 3 was EPR silent (SI, Figure S13a), and CSI-MS of 3 exhibited an ion peak at m/z of 947.2, corresponding to the mass and isotope distribution pattern of [(TPFC)Mn-(OH)(CH₃CN)₂]⁻ (calcd m/z = 947.0) (SI, Figure S13b). The spectroscopic data and the previous examples of Mn(III) corroles binding hydroxide, [(Corrole)Mn^III(OH)]⁻,²² led us to assign 3 as [(TPFC)Mn^III(OH)]⁻.

Then, electrochemical measurements of [(TPFC)Mn^III] were carried out in the absence and presence of OH⁻ (Figure 4). The cyclic voltammogram (CV) of [(TPFC)Mn^III] in CH₃CN revealed reversible anodic and cathodic peaks centered at 0.74 V vs SCE, which is assigned as a Mn^IV/III redox couple (Figure 4, red line). This value was similar to the previously reported redox potential of [(TPFC)Mn^III] in CH₂Cl₂ (0.71 V vs SCE).²³ Interestingly, when [(TPFC)Mn^III] was converted to [(TPFC)-Mn^III(OH)]⁻ by adding TMAH (2.0 mM) to the solution of [(TPFC)Mn^III] (2.0 mM), the Mn^IV/III redox potential exhibited a large negative shift from 0.74 V vs SCE in the absence of TMAH to 0.18 V vs SCE in the presence of TMAH (Figure 4, blue line). Because of the large negative shift of the first oxidation potential of [(TPFC)Mn^III(OH)]⁻, the second and third redox couples were observed at 0.45 and 0.71 V vs SCE, respectively. Thus, within the same region of the potential between +1.0 and –0.4 V for the oxidation of [(TPFC)Mn^III] in the absence of TMAH, up to three-electron oxidation of [(TPFC)Mn^III(OH)]⁻ becomes possible, although whether the second oxidation occurs at the metal center to afford the Mn^V complex or at the ligand to give the corrole radical cation has yet to be determined. Similar negative shift of the Mn^IV/III redox couple was obtained under an Ar atmosphere in THF. The redox potential was 0.89 V vs SCE in the absence of TMAH, which shifted negatively to 0.50 V vs SCE in the presence of TMAH (SI, Figure S14). The smaller negative shift in THF than that in CH₃CN may result from the weaker binding of OH⁻ of TMAH in THF.

Figure 4.

Cyclic voltammograms of [(TPFC)Mn^III] (2.0 mM) in the absence (red line) and presence (blue line) of TMAH (2.0 mM) in CH₃CN at 25 °C.

The Mn^IV/III redox couple of [(TPFC)Mn^III] was also shifted to the negative direction to 0.46 V vs SCE in the presence of excess Cl⁻ anion (SI, Figure S15, blue line). Such an anion effect on the redox potential of Mn corroles was reported by Kadish and co-workers;²² it has been demonstrated that Mn(III) corroles binding anions, such as Cl⁻, CN⁻, OAc⁻, F⁻, and OH⁻, changed their redox potentials with the negative shift of the redox couple of Mn^IV/III.22 Thus, Mn(III) corroles binding anionic axial ligands can be oxidized more easily as compared with those without axial ligands.

The cyclic voltammetry results corroborate the results of the O₂-activation reaction by [(TPFC)Mn^III]; the O₂-activation reaction by [(TPFC)Mn^III] occurs when the oxidation potential of [(TPFC)Mn^III] is low, such as in the case of OH⁻ binding (i.e., 0.18 V vs SCE). In other words, [(TPFC)Mn^III] binding OH⁻ can bind O₂ due to its low oxidation potential. In contrast, when the oxidation potential of [(TPFC)Mn^III] is relatively high, such as in the cases of no anionic axial ligand or with Cl⁻ anionic axial ligand, the Mn(III) corroles cannot bind O₂ and O₂-activation does not occur in these systems. On the basis of the electrochemical study of [(TPFC)Mn^III] in the absence and presence of anionic axial ligands, we conclude that the role of OH⁻ is to lower the oxidation potential of [(TPFC)Mn^III] and the [(TPFC)Mn^III] complex with an axially coordinated OH⁻ can bind O₂ to form a putative Mn(IV)-superoxo complex for further reactions (Scheme 2, pathways a and b). Indeed, it has been shown previously that the binding of O₂ becomes favorable when cobalt or manganese porphyrins bind imidazole or pyridine as an axial ligand²⁴ and the oxidation potential of a nonheme iron(II) complex becomes lower by changing a solvent system.²⁵

2.2.2. Role of THF and Cyclic Olefins in O₂-Activation Reaction

We have shown above that Mn-oxygen intermediates, 1 and 2, are formed when Mn(III) corroles are reacted with O₂ in the presence of base in THF, but not in CH₃CN (vide supra). Interestingly, in the latter reaction, addition of a small amount of THF to a CH₃CN solution containing [(TPFC)Mn^III] and TMAH (20 equiv) afforded the immediate formation of 2 at 25 °C (SI, Figure S16a). Similarly, addition of cyclic olefins (e.g., cyclopentene, cyclohexene, and cycloheptene) to a CH₃CN solution containing [(TPFC)Mn^III] (0.030 mM) and a large excess of TMAH (20 equiv, 0.60 mM) generated 2 immediately (SI, Figure S16b). We therefore investigated the role of THF and cyclic olefins in O₂-activation reaction, by carrying out the reaction of [(TPFC)Mn^III] with O₂ in CH₃CN at a lower temperature (e.g., at –20 °C) and by adding different amounts of H atom donors (e.g., THF and cyclic olefins) for kinetic studies. As described above, the mechanistic studies were performed using 20 equiv of TMAH, which was the condition for the generation of 2 with a clean kinetic behavior.

Addition of THF (0.12 M) to a reaction solution containing [(TPFC)Mn^III] (0.030 mM) and TMAH (20 equiv, 0.60 mM) at –20 °C afforded clean spectral changes with isosbestic points at 370, 407, 451, 498, 586, 666, and 740 nm for the formation of 2 (Figure 5a). The formation rate of 2 was dependent on the amounts of THF added; the first-order rate constants, determined by pseudo-first-order fitting of the kinetic data, increased linearly with the increase of the concentration of THF, affording a second-order rate constant of 6.1(5) × 10⁻² M⁻¹ s⁻¹ at –20 °C (SI, Figure S17). Similarly, when cyclic olefins, such as cyclohexene, cycloheptene and cyclopentene, were added to a CH₃CN solution containing [(TPFC)Mn^III] (0.030 mM) and TMAH (20 equiv, 0.60 mM) at –20 °C, we observed the formation of 2 with clean isosbestic points (Figure 5b). The formation of 2 was dependent on the concentration of olefins, and the second-order rate constants of 8.0(7) × 10⁻², 3.8(4) × 10⁻² and 1.9(2) × 10⁻² M⁻¹ s⁻¹ were determined in the reactions of cyclohexene, cycloheptene, and cyclopentene, respectively (Figure 5c; SI, Figures S18 and S19). However, no reaction occurred when cyclooctene was used. In addition, the formation of 2 was slower in the reaction of cyclohexene-d₁₀, giving a kinetic isotope effect (KIE) value of 5.0. We have also examined other substrates with weaker C–H bond other than cycloalkenes, such as fluorene. The second-order rate constant of fluorene was determined to be 2.9(3) × 10⁻¹ M⁻¹ s⁻¹ (SI, Figure S20), which was approximately 4 times larger than that of cyclohexene. These results indicate that the formation rate was correlated with the C–H bond dissociation energies (BDEs) of substrates (fluorene, BDE = 79.5 kcal mol⁻¹; cyclohexene, BDE = 81.0 kcal mol⁻¹; cycloheptene, BDE = 82.8 kcal mol⁻¹; cyclopentene, BDE = 83.3 kcal mol⁻¹ and cyclooctene, BDE = 85.4 kcal mol⁻¹), as observed in other metal complex-initiated O₂-activation reactions in the presence of cyclic olefins.^10d,26 No reaction with cyclooctene suggests that the BDE of the O–H bond of the Mn(IV)-hydroperoxo complex may be smaller than that of the C–H bond of cyclooctene (85.4 kcal mol⁻¹), when the H atom abstraction from cyclooctene by the Mn(IV)-hydroperoxo complex is uphill. The weaker O–H bond of the Mn(IV)-hydroperoxo complex than that of H₂O₂ (87.49 ± 0.07 kcal mol⁻¹)²⁷ may result from the conjugation of the Mn(IV)-superoxo radical complex, since it is known that the smaller the spin density of the terminal oxygen, the smaller the BDH of the O–H bond.²⁸ The linear correlation of log k_H, where the k_H values were obtained by dividing the second-order rate constants (k₂) by the numbers of equivalent target C–H bonds in the substrates (i.e., 4 for cycloalkenes and 2 for fiuorine), vs BDE is shown in Figure S21 in SI, where the Bell–Evans–Polanyi α value is determined as 0.42 ± 0.05.

Figure 5.

(a) UV–vis spectral changes for the formation of 2 (blue line) and the disappearance of 3 (red line) upon addition of THF (0.12 M) to a CH₃CN solution of [(TPFC)Mn^III(OH)]⁻ (3) (0.030 mM, red line) at –20 °C; 3 was prepared by adding TMAH (0.60 mM) to a CH₃CN solution of [(TPFC)Mn^III] (0.030 mM). Inset shows time course monitored at 431 nm due to 2. (b) UV–vis spectral changes for the formation of 2 (blue line) and the disappearance of 3 (red line) upon addition of cyclohexene (0.20 M) to a CH₃CN solution of [(TPFC)Mn^III(OH)]⁻ (3) (0.030 mM, red line) at –20 °C; 3 was prepared by adding TMAH (0.60 mM) to a CH₃CN solution of [(TPFC)Mn^III] (0.030 mM). Inset shows time course monitored at 431 nm due to 2. (c) Plots of k_obs against concentration of cyclohexene (black circles) and cyclohexene-d₁₀ (red rectangles) to determine the second-order rate constants for the formation of 2 in the O₂-activation by [(TPFC)Mn^III] (0.030 mM) in the presence of TMAH (0.60 mM) in air-saturated CH₃CN at –20 °C.

The results presented above demonstrate unambiguously that the rate of the formation of 2 depends on the BDEs of C–H bonds of cyclic olefins. However, it is of interest to note that although BDE of the C–H bond on α-position of THF is ~92 kcal mol⁻¹,²⁹ which is much higher than the BDE of the allylic C–H bonds of cyclohexene (81.0 kcal mol⁻¹), the rates of the formation of 2 in the reactions of THF (6.1 × 10⁻² M⁻¹ s⁻¹ at –20 °C) and cyclohexene (8.0 × 10⁻² M⁻¹ s⁻¹ at –20 °C) are similar. Indeed, such a unusual reactivity in the reaction of THF was previously reported in the H atom transfer reactions by tert-butoxyl radical³⁰ and a (μ- ν²: ν²-peroxo)dicopper(II) complex.³¹ In the latter case, the reactivity of THF was similar to the substrate with a BDE of ~82 kcal mol⁻¹, which is close to that of cyclohexene.³¹ The easy oxidation of THF by C–H bond activation was explained previously with a pronounced stereo-electronic effect on the H atom abstraction from those C–H bonds adjacent to the oxygen atom in THF.^31,32

On the basis of the experimental results presented above, it is evident that THF and cyclic olefins function as a H atom donor. In addition, no reaction occurred between 3 and THF or cycloalkenes under anaerobic conditions. Therefore, the H atom abstraction from the H atom donors (e.g., THF or cyclic olefins) by the putative Mn-superoxo species is the rate-determining step (r.d.s.) for the formation of Mn-oxygen intermediates in the O₂-activation reaction (Scheme 2, pathway c). It has been demonstrated recently that metal-superoxo species are capable of abstracting H atom from substrates to form metal-hydroperoxo species in enzymatic and biomimetic reactions.³³

2.2.3. Kinetic Studies of O₂-Binding and O–O Bond Formation

The concentration effect of O₂ in the dioxygen activation reaction was also investigated. The rate constant of the generation of 2 from 3 with cyclohexene in an O₂-saturated CH₃CN was determined to be 3.9(4) × 10 ¹ M⁻¹ s⁻¹ (SI, Figure S22), which is approximately 5 times larger than that in an air-saturated CH₃CN (8.0(7) × 10⁻² M⁻¹ s⁻¹). Thus, the equilibrium for formation of the Mn(IV)-superoxo complex lies far to the left-hand side, when the concentration of the Mn(IV)-superoxo complex is proportional to concentration of O₂ (Scheme 2, pathway b). As indicated by the deuterium kinetic isotope effect in the generation of 2 with cyclohexene-d₁₀ (KIE = 5.0), hydrogen atom transfer from cyclohexene to the Mn(IV)-superoxo species is the rate-determining step, which competes with the back reaction of the Mn(IV)-superoxo species to regenerate the Mn(III) complex. In such a case, the observed rate constant corresponds to k_HK, in which the equilibrium constant for the generation of the Mn(IV)-superoxo complex cannot be determined separately from the rate constant of hydrogen transfer (k_H).

Kinetic studies on the O–O bond formation between 1 and OH⁻ to produce 2 were examined as well. These experiments were performed at –40 °C, due to its fast conversion (SI, Figure S23). The rate of formation of 2 obeyed first-order kinetics and the pseudo-first-order rate constants were proportional to concentration of OH⁻. The second-order rate constant was determined from the slope of the linear plot to be 5.6(5) × 10 M⁻¹ s⁻¹. The linear dependence of the rate constant on OH⁻concentration also indicates that the nucleophilic attack of OH⁻to the Mn(V)-oxo species is the rate-determining step in the O–O bond formation, as proposed in literatures.^8a,b,34 Moreover, this rate constant (5.6(5) × 10 M⁻¹ s⁻¹ at –40 °C) is much larger than those of cycloalkenes in the O₂ activation to generate 2 (8.0(7) × 10⁻² M⁻¹ s⁻¹ for cyclohexene at –20 °C). This is fully consistent with the result that the rate-determining step in the O₂ activation to generate 2 is the H atom transfer from H atom donors to the Mn(IV)-superoxo species, followed by fast O–O bond formation to generate 2.

2.3. Proposed Mechanisms and Conclusions

The overall reaction mechanism for the formation of 1 and 2 in the O₂-activation reaction by [(TPFC)Mn^III] is depicted in Scheme 2, which shows (i) the formation of [(TPFC)-Mn^III(OH)]⁻ (3) upon addition of base to [(TPFC)Mn^III] (pathway a), (ii) the binding of O₂ by 3 to form a putative Mn^IV-superoxo species, [(TPFC)Mn^IV(O₂^.−)] (pathway b), (iii) the H atom abstraction from H atom donor by [(TPFC)Mn^IV(O₂^.−)] to form a putative Mn^IV-hydroperoxo species, [(TPFC)-Mn^IV(OOH)] (pathway c), (iv) the conversion of [(TPFC)-Mn^IV(OOH)] to [(TPFC)Mn^V(O)] via O–O bond homolysis (pathway d) or to [(TPFC)Mn^IV(O₂)]⁻ by deprotonation of the hydroperoxide ligand (pathway f), and (v) the conversion of 1 to 2 upon addition of OH⁻ to 1 (pathway e). We have also shown that (vi) addition of proton to 2 afforded 1 via the formation of [(TPFC)Mn^IV(OOH)] (pathway g), followed by the hydroperoxide O–O bond cleavage of the latter species (pathway d). The Mn(V)-oxo and Mn(IV)-peroxo intermediates were also synthesized independently by reacting [(TPFC)-Mn^III] with PhIO (pathway h) and H₂O₂/OH⁻ (pathway i), respectively, and were used for spectroscopic characterization and mechanistic studies. Detailed discussions for the reactions i–vi are presented below.

1. In the first step of the reaction cycle (Scheme 2, pathway a), the oxidation potential of [(TPFC)Mn^III] is shifted negatively by 0.56 V upon binding of OH⁻. Thus, the role of OH⁻ is to lower the oxidation potential of the Mn(III) corrole, which is then oxidized easily for further reaction.

2. The Mn(III) corrole (3) with a low oxidation potential can bind O₂ readily, thereby forming a putative Mn(IV)-superoxo species (Scheme 2, pathway b) via electron transfer from 3 to O₂, followed by binding of O₂ ^•− to the Mn(IV) center. Although electron transfer from 3 (E_ox = 0.18 V vs SCE) to O₂ (E_red = –0.87 V vs SCE) is still endergonic,³⁵ the binding of O₂ to the Mn(III) center may produce Mn(IV)-superoxo species via an inner-sphere electron transfer process, which is thermodynamically feasible. This step is probably in equilibrium, as shown in the binding of O₂ by many metal complexes (e.g., Co(II) complexes).^24,36 If there are no H atom donors, such as THF and cyclic olefins, no further reaction occurs and 3 is the only detectable species in reaction solution because the equilibrium is toward the dissociation of O₂ from the Mn(IV)-superoxo species.

3. In the presence of H atom donors, such as THF or cyclic olefins, the putative Mn(IV)-superoxo species abstracts a H atom from H atom donors, thereby forming a putative Mn(IV)-hydroperoxo species (Scheme 2, pathway c). We have verified that THF and cyclic olefins function as a H atom donor. In addition, based on the large KIE value determined in the reactions of cyclohexene and deuterated cyclohexene and the linear relationship between the BDEs of cyclic olefins and the formation rate constants of Mn-oxygen intermediates, H atom abstraction by the Mn(IV)-superoxo species was proposed to be the r.d.s.

4. In the reaction of using a smaller amount of base (e.g., 2 equiv to Mn corrole), Mn(V)-oxo species (1) was the product formed in the O₂-activation by [(TPFC)Mn^III] in the presence of a H atom donor (e.g., THF or cyclohexene). A proposed mechanism is the conversion of the putative Mn(IV)-hydroperoxo species to 1 via O–O bond homolysis (Scheme 2, pathway d). An alternative mechanism that can be considered is the O–O bond heterolysis of the Mn(IV)-hydroperoxo species to yield a highly unstable Mn(VI)-oxo species and OH⁻, followed by a fast one-electron reduction of the Mn(VI)-oxo species to form the Mn(V)-oxo species. The mechanisms of the hydroperoxide O–O bond cleavage of metal-hydroperoxo species has been extensively investigated in biomimetic studies.^5b,37

5. In the case of using a larger amount of base (e.g., 20 equiv to Mn corrole), Mn(IV)-peroxo species (2) instead of Mn(V)-oxo (1) was formed as the product in the O₂-activation reaction by [(TPFC)Mn^III]. As proposed in Scheme 2, 2 can be formed either from the reaction of 1 and OH⁻ (pathway e) or from the deprotonation of the Mn(IV)-hydroperoxo species (pathway f). The former pathway, which shows the O–O bond formation between Mn(V)-oxo and OH⁻, has been the topic of current research for the O₂-evolution process in water oxidation by PS II. It is of interest to note that despite the intense research with a large number of high-valent metal-oxo species aimed at understanding the O–O bond formation step in model studies of PS II, such an O–O bond formation has only been directly observed with Mn(V)–O corrole species.¹² The nucleophilic attack of OH⁻ to 1 might produce [(TPFC)Mn^III(OOH)]⁻ complex and the latter species was then oxidized by 1 to form [(TPFC)-Mn^IV(OOH)]⁻ intermediate, followed by the deprotona-tion by OH⁻ to afford 2 as the final product. However, detailed mechanism for the O–O bond formation between high-valent metal-oxo species and OH⁻ remains elusive.

   Another plausible pathway for the formation of 2 is the deprotonation of the putative Mn(IV)-hydroperoxo species (pathway f), which has been well documented in the acid–base chemistry between metal-hydroperoxo and -peroxo species in biomimetic reactions.^17c,37c,38 As mentioned above, the formation mechanism of 2, such as the O–O bond formation between 1 and OH⁻ or the deprotonation of the Mn(IV)-hydroperoxo species (pathway f), cannot be distinguished without capturing the Mn(IV)-OOH species and determining the rates of the O–O bond cleavage and deprotonation steps of the putative Mn(IV)-OOH species.

6. The reverse reaction of the O–O bond formation between 1 and OH⁻ is the protonation of 2 that leads to the formation of the Mn(IV)-hydroperoxo species (pathway g), followed by the O–O bond cleavage of the Mn(IV)-hydroperoxo species to form 1 (pathway d). It has been well documented in biomimetic studies that protonation of metal-peroxo species generates high-valent metal-oxo species via the formation of metal-hydroperoxo species, followed by either homolysis or heterolysis of the hydroperoxide O–O bond of the metal-hydroperoxo species.^5b,37

In summary, we have shown that Mn(III) corroles are able to activate O₂ in the presence of base and H atom donor, thereby forming Mn(V)-oxo and Mn(IV)-peroxo species. We have also shown the reversibility of the O–O bond formation and cleavage reactions between Mn(V)-oxo and Mn(IV)-peroxo species. To the best of our knowledge, the present study reports the first example of using the same manganese complex in both O₂-activation and O–O bond formation reactions.

3. EXPERIMENTAL SECTION

3.1. Materials

Commercially available chemicals were used without further purification unless otherwise indicated. Solvents were dried according to the literature procedures and redistilled under Ar before use.³⁹ 1-(tert-butylsulfonyl)-2-iodosylbenzene (^sPhIO) was synthesized by a literature method.⁴⁰ The purity of the oxidant was determined by iodometric titration. [(TPFC)Mn^III] and [(TPFcCN)Mn^III] were synthesized by following the literature.^{41 18}O₂ (98% ¹⁸O-enriched) and H₂¹⁸O (95% ¹⁸O-enriched) were purchased from ICON Services Inc. (Summit, NJ, USA).

3.2. Instrumentation

UV–vis spectra were recorded on a Hewlett-Packard Agilent 8453 UV–vis spectrophotometer equipped with an UNISOKU cryostat system (USP-203; UNISOKU, Japan). Coldspray ionization time-of-flight mass (CSI-MS) spectral data were collected on a JMS-T100CS (JEOL) mass spectrometer equipped with a CSI source. Typical measurement conditions were as follows: needle voltage, 2.2 kV; orifice 1 current, 50–500 nA; orifice 1 voltage, 0 to 20 V; ring lens voltage, 10 V; ion source temperature, 5 °C; spray temperature, –40 °C. Resonance Raman scattering was dispersed by a single polychromator (Ritsu Oyo Kogaku, MC-100DG) and was detected by a liquid-nitrogen-cooled CCD detector (HORIBA JOBIN YVON, Symphony 1024 × 128 Cryogenic Front Illuminated CCD Detector). Raman spectra were collected with backscattering geometry at an excitation wavelength (λ_ex) of 405 nm (LM-405-PLR-40-2, ONDAX) using a spinning sample cell (NMR tube with 5 mm OD), which was placed in a thermostated quarts Dewar at –40 °C by flashing cold nitrogen gas. The laser power at a measuring point in front of a quartz Dewar was adjusted to 20 mW. Raman shifts were calibrated using indene (accurate to within ±1 cm⁻¹). X-band electron paramagnetic resonance (EPR) spectra were recorded at 5 K using an X-band Bruker EMX-plus spectrometer equipped with a dual mode cavity (ER 4116DM). Low temperatures were achieved and controlled with an Oxford Instruments ESR900 liquid Helium quartz cryostat fitted with an Oxford Instruments ITC503 temperature and gas flow controller. The experimental parameters for EPR spectra were as follows: Microwave frequency = 9.647 GHz, microwave power = 1.0 mW, modulation amplitude = 10 G, gain = 1 × 10⁴, modulation frequency = 100 kHz, time constant = 40.96 ms, and conversion time = 81.00 ms. Electrochemical measurements were performed on a CHI630B electrochemical analyzer (CH Instruments, Inc.) in deaerated CH₃CN containing 0.10 M Bu₄NPF₆ (TBAPF₆) as a supporting electrolyte at 25 °C. A conventional three-electrode cell was used with a platinum working electrode (surface area of 0.30 mm²), a platinum wire as a counter electrode, and an Ag/Ag⁺ electrode as a reference electrode. The platinum working electrode was routinely polished with BAS polishing alumina suspension and rinsed with acetone and acetonitrile before use. The measured potentials were recorded with respect to an Ag/Ag⁺ (0.010 M) reference electrode. All potentials (vs Ag/Ag⁺) were converted to values vs SCE by adding 0.29 V.

3.3. X-ray Absorption Spectroscopy

The Mn K-edge X-ray absorption spectra of [(TPFC)Mn^III] and 2 were measured at the Stanford Synchrotron Radiation Lightsource (SSRL) on the wiggler BL9-3 under standard ring conditions of 3 GeV and ~500 mA. A Si(220) double crystal monochromator was used for energy selection. A Rh-coated harmonic rejection mirror was used to reject components of higher harmonics. During data collection, the samples were maintained at a constant temperature of ~10 K using an Oxford Instruments CF 1208 liquid helium cryostat. A 100-element Ge monolith fluorescence detector from Canberra Industries was employed for fluorescence data measurement to k = 12 Å⁻¹. Solution samples for [(TPFC)Mn^III] and intermediate 2 were transferred into 2 mm delrin XAS cells with 70 m Kapton tape windows under synthesis conditions and were immediately frozen after preparation and stored under liquid N₂ conditions. Internal energy calibration was accomplished by simultaneous measurement of the absorption of a Mn-foil placed between two ionization chambers situated after the sample. The first inflection point of the foil spectrum was set at 6539.0 eV. Data presented here are 8-scan average spectra for [(TPFC)Mn^III] and 16-scan average spectrum for 2. To avoid potential photodamage, at most two scans were obtained on a fresh sample. Significant photoreduction was not observed (judging by comparison of scan 1 and scan 2). Data were processed by fitting a second-order polynomial to the pre-edge region and then subtracting this from the entire spectrum as background. A three-region spline of orders 2, 3, and 3 was used to model the smoothly decaying postedge region. The data were normalized by subtracting the cubic spline and assigning the edge jump to 1.0 at 6555 eV using the Pyspline⁴² program. Data were then renormalized in Kaleidagraph for comparison and quantitation purposes. Theoretical EXAFS signals χ (k) were calculated by using FEFF (Macintosh version)^43–45 using geometry optimized preliminary structures obtained from DFT calculations. The input structure was improved based on preliminary EXAFS fit parameters to generate more accurate theoretical EXAFS signals. Data fitting was performed in EXAFSPAK.⁴⁶ The structural parameters varied during the fitting process were the bond distance (R) and the bond variance σ², which is related to the Debye–Waller factor resulting from thermal motion, and static disorder of the absorbing and scattering atoms. The nonstructural parameter E₀ (the energy at which k = 0) was also allowed to vary but was restricted to a common value for every component in a given fit. Coordination numbers was systematically varied in the course of the fit but were fixed within a given fit.

3.4. Generation of Intermediate 1

[(TPFC)Mn^V(O)] (1) intermediate was generated by adding tetramethylammonium hydroxide (TMAH, 2 equiv, 0.060 mM) into a UV–vis cuvette containing a THF solution of [(TPFC)Mn^III] (0.030 mM) under air atmosphere at 25 °C. Formation of 1 was confirmed by monitoring UV–vis spectral changes at 478 and 598 nm due to the decay of [(TPFC)Mn^III] and at 347 and 407 nm due to the formation of 1. The ¹⁸O-labeled complex [(TPFC)Mn^V(¹⁸O)] (1-¹⁸O) was also generated by adding tetrame-thylammonium hydroxide (TMAH, 2 equiv, 0.060 mM) into a UV–vis cuvette containing a THF solution of [(TPFC)Mn^III] (0.030 mM) under ¹⁸O₂ atmosphere at 25 °C.

1 can also be generated by adding ^sPhIO (2 equiv) into a CH₃CN solution of [(TPFC)Mn^III] (0.030 mM) at 0 °C. 1 was generated within 1 min. [(TPFcCN)Mn^V(O)] intermediate was generated by the same procedure for the generation of 1.

UV–vis of 1 in THF, λ_max (nm) (ε, M⁻¹ cm⁻¹): 347 (4.5 × 10⁴), 407 (4.0 × 10⁴), 520 (7.6 × 10³). UV–vis of 1 in CH₃CN: 347 (4.6 × 10⁴), 405 (4.2 × 10⁴), 518 (8.4 × 10³).

3.5. Generation of Intermediate 2

[(TPFC)Mn^IV(O₂)]⁻ (2) complex was generated by adding tetramethylammonium hydroxide (TMAH, 20 equiv, 0.60 mM) into a UV–vis cuvette containing a THF solution of [(TPFC)Mn^III] (0.030 mM) under air atmosphere at 25 °C. 2 was also generated by adding tetramethylammonium hydroxide (TMAH, 20 equiv, 0.60 mM) into a UV–vis cuvette containing a CH₃CN solution of [(TPFC)Mn^III] (0.030 mM) in the presence of THF or cyclic olefins, such as cyclohexene and cyclopentene, under air atmosphere at 25 °C.

Alternatively, 2 can be prepared by reacting [(TPFC)Mn^III] (0.030 mM) with H₂O₂ solution (0.12 mM) in the presence of TMAH (0.60 mM, 20 equiv) in CH₃CN at 0 °C. [(TPFcCN)Mn^IV(O₂)]⁻ was generated by the same procedure for the generation of 2.

UV–vis of 2 in THF, λ_max (nm) (ε, M⁻¹ cm⁻¹): 435 (5.7 × 10⁴), 574 (1.3 × 10⁴), 696 (3.5 × 10³). UV–vis of 2 in CH₃CN: 431 (5.3 × 10⁴), 571 (1.3 × 10⁴), 701 (3.2 × 10³).

3.6. Reversible Transformation between 1 and 2. 1

(0.030 mM), which was generated by dioxygen activation from [(TPFC)-Mn^III] and TMAH under air atmosphere, was converted to 2 immediately by adding TMAH (20 equiv) into a THF solution of 1 at 25 °C. The same conversion was also obtained by using 1 produced in the reaction of [(TPFC)Mn^III] and ^sPhIO.

To the opposite, 2 (0.030 mM), which was generated by dioxygen activation from [(TPFC)Mn^III] and TMAH, was also reversibly converted to 1 by adding HClO₄ (0.60 mM, 20 equiv) into a THF solution of 2 at 25 °C. The same conversion was also obtained by using 2 produced in the reaction of [(TPFC)Mn^III] and H₂O₂ in the presence of TMAH.

3.7. Generation of Intermediate 3

[(TPFC)Mn^III(OH)]⁻ was produced by adding TMAH (20 equiv) into a CH₃CN solution of [(TPFC)Mn^III] (0.030 mM) under air at 25 °C. No further spectral change was observed upon addition of more than 20 equiv of TMAH.

UV–vis of 3 (CH₃CN), λ_max (nm) (ε, M⁻¹ cm⁻¹): 401 (3.7 × 10⁴), 428 (4.0 × 10⁴), 471 (4.7 × 10³), 562 (9.5 × 10³), 595 (9.0 × 10³), 636 (1.1 × 10⁴).

3.8. Kinetic Studies

All reactions were investigated in a 1.0 cm quartz cuvette and followed by monitoring UV–vis spectral changes of the generation of 2 in CH₃CN/THF or CH₃CN/cyclic olefins at –20 °C. The kinetic experiments were run at least in triplicate, and the data reported here represented the average of these reactions. Rate constants were determined under pseudo-first-order conditions (i.e., [substrate]/ [intermediate] > 10) by fitting the changes in absorbance at 431 nm due to the formation of 2. Kinetic isotope efiect was determined by comparing the formation rates of 2 in the presence of cyclohexene and cyclohexene-d₁₀.