Formation of a Reactive, Alkyl Thiolate-Ligated Fe^III-Superoxo Intermediate Derived from Dioxygen

Abstract

Herein, we describe an alkyl thiolate-ligated iron complex that reacts with dioxygen to form an unprecedented example of an iron superoxo (O₂^•−) intermediate, [Fe^III(S₂^Me2N₃(Pr,Pr))(O₂)] (4), which is capable of cleaving strong C–H bonds. A cysteinate-ligated iron superoxo intermediate is proposed to play a key role in the biosynthesis of β-lactam antibiotics by isopenicillin N-synthase (IPNS). Superoxo 4 converts to a metastable putative Fe(III)–OOH intermediate, at rates that are dependent on the C–H bond strength of the H atom donor, with a kinetic isotope effect (k_H/k_D = 4.8) comparable to that of IPNS (k_H/k_D = 5.6). The bond dissociation energy of the C–H bonds cleaved by 4 (92 kcal/mol) is comparable to C–H bonds cleaved by IPNS (93 kcal/mol). Both the calculated and experimental electronic absorption spectra of 4 are comparable to those of the putative IPNS superoxo intermediate, and are shown to involve RS⁻ → Fe–O₂^•− and O₂^•− → Fe charge transfer transitions. The π-back-donation by the electronrich alkyl thiolate presumably facilitates this reactivity by increasing the basicity of the distal oxygen. The frontier orbitals of 4 are shown to consist of two strongly coupled unpaired electrons of opposite spin, one in a superoxo π*(O–O) orbital, and the other in an Fe(d_xy) orbital.

Isopenicillin N synthase (IPNS)^1–4 and cysteine dioxygenase (CDO)^5–10 are nonheme Fe enzymes that catalyze the O₂-promoted oxidation of cysteinates (RS⁻). Although O₂ oxidation reactions are thermodynamically favored, they are kinetically slow in the absence of a transition-metal catalyst, because they are spin-forbidden.¹¹ The electron donor properties of cysteinate and high covalency of Fe^III–SR bonds^12,13 lower the activation barrier to O₂ binding⁴ to iron, promote O–O bond cleavage,^4,14 and increase the reactivity of high-valent Fe-oxo intermediates.^15,16 This helps to facilitate the oxidative bicyclization reaction involved in the biosynthesis of β-lactam antibiotics (e.g., penicillin, cephalosporins) by IPNS,^1,3 as well as the CDO-catalyzed regulation of cysteine concentration, toxic levels of which can lead to neurological disorders,¹⁷ or the metastasis of cancerous tumors.^18,19 The proposed mechanism of both IPNS^2,3 and CDO^5,6 involves the initial formation of a cis thiolate-ligated iron superoxo intermediate (cis-RS-Fe-O₂^•−). With the former, this intermediate abstracts a H atom from substrate, and with the latter it is proposed to attack the adjacent sulfur to form a transient peroxythiolate species. The putative IPNS Fe–O₂^•− is spectroscopically detected in small amounts (14%) via transient absorption (λ_max = 630 nm; t = 2–10 ms) and Mössbauer spectroscopies if the cysteinate β-hydrogens are deuterated.² A CDO intermediate, proposed to be an Fe(III)-peroxythiolate, is also observed by transient absorption spectroscopy (λ_max = 500 nm, 640 nm).⁵ Vibrational data is not available to support these assignments, however. Two strong C–H bonds are cleaved during the proposed IPNS mechanism (Figure S1): a β-hydrogen from cysteine (93 kcal/mol) and a β-hydrogen from valine (96 kcal/mol).²⁰ The former is proposed to involve the putative Fe(III)-superoxo, and the latter an Fe(IV)-oxo intermediate.² There are few well-characterized examples of Fe(III)-superoxo compounds,^21–24 however, and none of these cleave strong C–H bonds. An aryl thiolate-ligated Fe–O₂^•− was recently reported; however, the sulfur lone pair is tied up in π-bonding to the aryl carbon in one of its resonance forms making it less reactive.²³ Although it has yet to be demonstrated with a superoxo, π-back-donation by an electron rich alkyl thiolate has been shown to facilitate the cleavage of strong C–H bonds by increasing the basicity of an iron oxo.²⁵ Herein, we report the synthesis and structure of an alkyl thiolate-ligated iron complex that reacts with O₂ to afford a spectroscopically observable reactive intermediate.

Reduced [Fe^II(S₂^Me2N₃(Pr,Pr))] (1) was synthesized and structurally characterized according to the method outlined in the Supporting Information, and was shown to contain Fe²⁺ in a distorted trigonal bipyramidal coordination environment (τ = 0.78; Figure 1, Tables S2–S6). In solution, 1 has a magnetic moment of μ = 2.63 μ_B at 298 K in MeCN consistent with an S = 1 spin-state, and has a characteristic electronic absorption band at λ_max = 420 (ε_M 1600) nm (Figure S2). Previously, we showed that, like IPNS and CDO,^3,10,26 the oxidized derivative of 1, [Fe^III(S₂^Me2N₃(Pr,Pr))]⁺ (2), binds small molecules (azide and NO),^27–29 cis with respect to one of the thiolate sulfurs. The latter are frequently used to probe enzymatic O₂ binding sites_.^10,14

Figure 1.

ORTEP of 1 with thermal ellipsoids at the 50% probability level. With the exception of the secondary amine proton, hydrogens have been removed for clarity.

The addition of dry O₂ to 1 in THF at 25 °C causes an immediate color change from pale yellow to watermelon pink, with an associated shift in λ_max to 510(1500) nm (Figure S3), and the growth of a signal (g = 2.17, 2.11, 1.98) in the electron paramagnetic resonance (EPR) spectrum (Figure S4) consistent with the formation (in 93% yield) of low-spin (S = ½) [Fe^III(η²-S^Me2O)(S^Me2N₃(Pr,Pr))]⁺ (3).³⁰ Electrospray mass spectroscopy (ESI-MS) of isotopically labeled samples shows that the oxo of 3 is derived from ¹⁸O₂ (Figure S5). Azide inhibits this reaction (Figures S6) indicating that O₂ must bind to the metal ion in order for oxo atom transfer to occur. At low temperatures (−73 °C), a new metastable cranberry red O₂-derived intermediate, 4 (Figure 2), is observed en route to singly oxygenated 3,³⁰ the low energy band (~700 nm) of which is characteristic of six-coordinate, bis-thiolate-ligated Fe(III).^12,31 When this reaction is monitored by ¹H NMR, the paramagnetic signals of 1 collapse to diamagnetic (S = 0) signals upon the addition of O₂ (Figure S7). The ESI-MS of 4 contains an M + 32 peak at m/z = 417.3 (Figure S8), consistent with the addition of two oxygen atoms to the parent ion, 1 (m/z = 385.4). An identical intermediate can be generated via the low temperature (−73 °C) addition of excess (50 equiv) potassium superoxide (KO₂) to oxidized [Fe^III(S₂^Me2N₃(Pr,Pr))]⁺ (2) (Figure S9). The resonance Raman (rR) spectrum of 4 reproducibly shows isotopically sensitive features (a Fermi doublet) at 1093 and 1122 cm⁻¹ (Figure 3) that shift to 1022 cm⁻¹ when generated from ¹⁸O₂ (Figure S10), and disappear after 30 min, demonstrating its transient nature. All of this data would be consistent with the formation of a metastable ferric superoxo species. The calibrated (vide infra) density functional theory (DFT) calculated structure of [Fe^III(S₂^Me2N₃(Pr,Pr))(O₂)] (4) contains an O₂ moiety cis to one of the thiolate sulfurs (Figure S11), with bond lengths (O–O = 1.289 Å, Table S1), and a calculated ν_O–O stretch (Figure S12, 1154 cm⁻¹), consistent with a ferric superoxo (Fe^III-O₂^•−), analogous to the prop osed IPNS and CDO intermediates. The frontier orbitals of 4 (Figure 4) contain two unpaired electrons of opposite spin, one in a superoxo π*(O–O) orbital, and the other in a Fe(d_xy) orbital. The calculated overlap parameter of T=0.17, and coupling constant J^calc = − 450 cm⁻¹ indicate that the two unpaired spins are strongly coupled antiferromagnetically, consistent with the absence of paramagnetically shifted peaks in the ¹H NMR and EPR silence of 4. The time-dependent DFT (TD-DFT) calculated electronic absorption spectrum of 4 (Figures S13 and S14) reproduces the experimental spectrum (Figure 2), and shows that superoxo π*(O–O) → d_xy(Fe) charge transfer transitions are responsible for the higher energy bands, and a RS⁻ → Fe–O₂^•− charge transfer transition for the lower energy band. Both the calculated and experimental spectrum of 4 are similar to that of the putative IPNS superoxo intermediate,² supporting its assignment as a superoxo species. The reported CDO intermediate spectrum⁵ is also similar to that of 4, suggesting that it too is a ferric superoxo.

Figure 2.

Monitoring the low temperature (−73 °C) reaction between 1 (0.48 mM) and excess O₂ in THF by electronic absorption spectroscopy.

Figure 3.

Monitoring the reaction between 1 (5 mM) and O₂ in THF at −73 °C by resonance Raman spectroscopy. Samples were frozen in liquid N₂ (77 K) at the time-intervals indicated. Excitation wavelength λ_ex = 527 nm; 4.0 mW power; * = solvent peak.

Figure 4.

Singly occupied molecular orbitals (SOMO) of 4 contain strongly coupled electrons of opposite spin, one on the superoxo (O₂^•−) and the other on the metal ion.

Ferric superoxo (Fe^III−O₂^•−) 4 converts to a second metastable intermediate, 5 (λ_max = 696 nm), at −73 °C in THF (Figure S15), en route to 3 (Figure S16), at a rate that is dependent on the C–H bond strength of the solvent or H atom donor. Reaction rates decrease in deuterated THF (Figure 5), and increase upon the addition of a sacrificial H atom donor (100 equiv of 1,4-cyclohexadiene (CHD), BDE = 76 kcal/mol). The observed deuterium isotope effect, k_H/k_D = 4.8, is comparable to that of IPNS (k_H/k_D = 5.6),³² and indicates that superoxo 4 is capable of abstracting hydrogen atoms from strong C–H bonds (BDE(THF) = 92 kcal/mol).³³ A likely product of this reaction would be a ferric hydroperoxo, [Fe^III(S₂ ^Me2N₃(Pr,Pr))(OOH)] (5). Consistent with this, a new rhombic signal grows in when the reaction between 1 and O₂ is monitored by EPR (Figure 6). Spin-quantitation using double integration (Figure S18) indicates that the EPR signal of 5 represents 87% of the sample (Figures S4 and S17). The remaining 13% can be attributed to 1 and/or 4, both of which are EPR-silent in ⊥-mode. Together, these results show that in contrast to the few reported Fe(III)-superoxo complexes,^21–24 alkylthiolate-ligated 4 is capable of abstracting H atoms from strong C–H bonds, on par with that of the β C–H bonds of cysteine (93 kcal/mol).³³ It is plausible that π-back-donation by the electron-rich alkyl thiolate facilitates this reactivity by increasing the basicity of the distal oxygen. Spectroscopic characterization of 4, along with calibrated DFT calculations, provides additional evidence to support the assignment of the IPNS and CDO intermediates detected via transient absorption spectroscopy,^2,5 as cis RS-Fe^III-O₂^•− species.

Figure 5.

Pseudo-first-order kinetic plots associated with the reaction between 4 (0.48 mM) and THF (12 M), or CHD (48 mM) in THF at −73 °C.

Figure 6.

X-band EPR spectrum (⊥-mode) of putative hydroperoxo Fe^III–OOH (5), formed from superoxo Fe^III–O₂^•− (4) via H atom abstraction.