Repurposing Nonheme Iron Hydroxylases To Enable Catalytic Nitrile Installation through an Azido Group Assistance

Three mononuclear nonheme iron and 2-oxoglutarate dependent enzymes, l-Ile 4-hydroxylase, l-Leu 5-hydroxylase and polyoxin dihydroxylase, are previously reported to catalyze the hydroxylation of l-isoleucine, l-leucine, and l-α-amino-δ-carbamoylhydroxyvaleric acid (ACV). In this study, we showed that these enzymes can accommodate leucine isomers and catalyze regiospecific hydroxylation. On the basis of these results, as a proof-of-concept, we demonstrated that the outcome of the reaction can be redirected by installation of an assisting group within the substrate. Specifically, instead of canonical hydroxylation, these enzymes can catalyze non-native nitrile group installation when an azido group is introduced. The reaction is likely to proceed through CH bond activation by an Fe­(IV)-oxo species, followed by azido-directed CN bond formation. These results offer a unique opportunity to investigate and expand the reaction repertoire of Fe/2OG enzymes

Modeling Non-Heme Iron Halogenases: High-Spin Oxoiron(IV)–Halide Complexes That Halogenate C–H Bonds

The non-heme iron halogenases CytC3 and SyrB2 catalyze C–H bond halogenation in the biosynthesis of some natural products via <i>S</i> = 2 oxoiron­(IV)–halide intermediates. These oxidants abstract a hydrogen atom from a substrate C–H bond to generate an alkyl radical that reacts with the bound halide to form a C–X bond chemoselectively. The origin of this selectivity has been explored in biological systems but has not yet been investigated with synthetic models. Here we report the characterization of <i>S</i> = 2 [Fe^IV(O)­(TQA)­(Cl/Br)]⁺ (TQA = tris­(quinolyl-2-methyl)­amine) complexes that can preferentially halogenate cyclohexane. These are the first synthetic oxoiron­(IV)–halide complexes that serve as spectroscopic and functional models for the halogenase intermediates. Interestingly, the nascent substrate radicals generated by these synthetic complexes are not as short-lived as those obtained from heme-based oxidants and can be intercepted by O₂ to prevent halogenation, supporting an emerging notion that rapid rebound may not necessarily occur in non-heme oxoiron­(IV) oxidations

Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI

The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH₂-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase CmlI. Upon exposure of the diferrous cluster to O₂, CmlI forms a long-lived peroxo intermediate, <b>P</b>, which reacts with NH₂-CAM to form CAM. Since <b>P</b> is capable of at most a two-electron oxidation, the overall reaction must occur in several steps. It is unknown whether <b>P</b> is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under ¹⁸O₂ show that both oxygen atoms in the nitro function of CAM derive from O₂. However, when the single-turnover reaction between ¹⁸O₂-<b>P</b> and NH₂-CAM is carried out in an ¹⁶O₂ atmosphere, CAM nitro groups contain both ¹⁸O and ¹⁶O, suggesting that <b>P</b> can be reformed during the reaction sequence. Such reformation would require reduction by a pathway intermediate, shown here to be NH­(OH)-CAM. Accordingly, the aerobic reaction of NH­(OH)-CAM with diferric CmlI yields <b>P</b> and then CAM without an external reductant. A catalytic cycle is proposed in which NH₂-CAM reacts with <b>P</b> to form NH­(OH)-CAM and diferric CmlI. Then the NH­(OH)-CAM rereduces the enzyme diiron cluster, allowing <b>P</b> to reform upon O₂ binding, while itself being oxidized to NO-CAM. Finally, the reformed <b>P</b> oxidizes NO-CAM to CAM with incorporation of a second O₂-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site

Characterization of the Fleeting Hydroxoiron(III) Complex of the Pentadentate TMC-py Ligand

Nonheme mononuclear hydroxoiron­(III) species are important intermediates in biological oxidations, but well-characterized examples of synthetic complexes are scarce due to their instability or tendency to form μ-oxodiiron­(III) complexes, which are the thermodynamic sink for such chemistry. Herein, we report the successful stabilization and characterization of a mononuclear hydroxoiron­(III) complex, [Fe^III(OH)­(TMC-py)]²⁺ (<b>3</b>; TMC-py = 1<i>-</i>(pyridyl-2′-methyl)-4,8,11-trimethyl-1,4,8,11-tetrazacyclotetradecane), which is directly generated from the reaction of [Fe^IV(O)­(TMC-py)]²⁺ (<b>2</b>) with 1,4-cyclohexadiene at −40 °C by H-atom abstraction. Complex <b>3</b> exhibits a UV spectrum with a λ_max at 335 nm (ε ≈ 3500 M^–1 cm^–1) and a molecular ion in its electrospray ionization mass spectrum at <i>m</i>/<i>z</i> 555 with an isotope distribution pattern consistent with its formulation. Electron paramagnetic resonance and Mössbauer spectroscopy show <b>3</b> to be a high-spin Fe­(III) center that is formed in 85% yield. Extended X-ray absorption fine structure analysis reveals an Fe–OH bond distance of 1.84 Å, which is also found in [(TMC-py)­Fe^III–O–Cr^III(OTf)₃]⁺ (<b>4</b>) obtained from the reaction of <b>2</b> with Cr­(OTf)₂. The <i>S</i> = 5/2 spin ground state and the 1.84 Å Fe–OH bond distance are supported computationally. Complex <b>3</b> reacts with 1-hydroxy-2,2,6,6-tetramethylpiperidine (TEMPOH) at −40 °C with a second-order rate constant of 7.1 M^–1 s^–1 and an OH/OD kinetic isotope effect value of 6. On the basis of density functional theory calculations, the reaction between <b>3</b> and TEMPOH is classified as a proton-coupled electron transfer as opposed to a hydrogen-atom transfer

Spectroscopic and Reactivity Comparisons between Nonheme Oxoiron(IV) and Oxoiron(V) Species Bearing the Same Ancillary Ligand

This work directly compares the spectroscopic and reactivity properties of an oxoiron­(IV) and an oxoiron­(V) complex that are supported by the same neutral tetradentate N-based PyNMe3 ligand. A complete spectroscopic characterization of the oxoiron­(IV) species (2) reveals that this compound exists as a mixture of two isomers. The reactivity of the thermodynamically more stable oxoiron­(IV) isomer (2b) is directly compared to that exhibited by the previously reported 1e–-oxidized analogue [FeV(O)­(OAc)­(PyNMe3)]2+ (3). Our data indicates that 2b is 4 to 5 orders of magnitude slower than 3 in hydrogen atom transfer (HAT) from C–H bonds. The origin of this huge difference lies in the strength of the O–H bond formed after HAT by the oxoiron unit, the O–H bond derived from 3 being about 20 kcal·mol–1 stronger than that from 2b. The estimated bond strength of the FeIVO–H bond of 100 kcal·mol–1 is very close to the reported values for highly active synthetic models of compound I of cytochrome P450. In addition, this comparative study provides direct experimental evidence that the lifetime of the carbon-centered radical that forms after the initial HAT by the high valent oxoiron complex depends on the oxidation state of the nascent Fe–OH complex. Complex 2b generates long-lived carbon-centered radicals that freely diffuse in solution, while 3 generates short-lived caged radicals that rapidly form product C–OH bonds, so only 3 engages in stereoretentive hydroxylation reactions. Thus, the oxidation state of the iron center modulates not only the rate of HAT but also the rate of ligand rebound

Spectroscopic Description of the E₁ State of Mo Nitrogenase Based on Mo and Fe X‑ray Absorption and Mössbauer Studies

Mo nitrogenase (N2ase) utilizes a two-component protein system, the catalytic MoFe and its electron-transfer partner FeP, to reduce atmospheric dinitrogen (N2) to ammonia (NH3). The FeMo cofactor contained in the MoFe protein serves as the catalytic center for this reaction and has long inspired model chemistry oriented toward activating N2. This field of chemistry has relied heavily on the detailed characterization of how Mo N2ase accomplishes this feat. Understanding the reaction mechanism of Mo N2ase itself has presented one of the most challenging problems in bioinorganic chemistry because of the ephemeral nature of its catalytic intermediates, which are difficult, if not impossible, to singly isolate. This is further exacerbated by the near necessity of FeP to reduce native MoFe, rendering most traditional means of selective reduction inept. We have now investigated the first fundamental intermediate of the MoFe catalytic cycle, E1, as prepared both by low-flux turnover and radiolytic cryoreduction, using a combination of Mo Kα high-energy-resolution fluorescence detection and Fe K-edge partial-fluorescence-yield X-ray absorption spectroscopy techniques. The results demonstrate that the formation of this state is the result of an Fe-centered reduction and that Mo remains redox-innocent. Furthermore, using Fe X-ray absorption and 57Fe Mössbauer spectroscopies, we correlate a previously reported unique species formed under cryoreducing conditions to the natively formed E1 state through annealing, demonstrating the viability of cryoreduction in studying the catalytic intermediates of MoFe

Spectroscopic and Reactivity Comparisons between Nonheme Oxoiron(IV) and Oxoiron(V) Species Bearing the Same Ancillary Ligand

This work directly compares the spectroscopic and reactivity properties of an oxoiron­(IV) and an oxoiron­(V) complex that are supported by the same neutral tetradentate N-based PyNMe3 ligand. A complete spectroscopic characterization of the oxoiron­(IV) species (2) reveals that this compound exists as a mixture of two isomers. The reactivity of the thermodynamically more stable oxoiron­(IV) isomer (2b) is directly compared to that exhibited by the previously reported 1e–-oxidized analogue [FeV(O)­(OAc)­(PyNMe3)]2+ (3). Our data indicates that 2b is 4 to 5 orders of magnitude slower than 3 in hydrogen atom transfer (HAT) from C–H bonds. The origin of this huge difference lies in the strength of the O–H bond formed after HAT by the oxoiron unit, the O–H bond derived from 3 being about 20 kcal·mol–1 stronger than that from 2b. The estimated bond strength of the FeIVO–H bond of 100 kcal·mol–1 is very close to the reported values for highly active synthetic models of compound I of cytochrome P450. In addition, this comparative study provides direct experimental evidence that the lifetime of the carbon-centered radical that forms after the initial HAT by the high valent oxoiron complex depends on the oxidation state of the nascent Fe–OH complex. Complex 2b generates long-lived carbon-centered radicals that freely diffuse in solution, while 3 generates short-lived caged radicals that rapidly form product C–OH bonds, so only 3 engages in stereoretentive hydroxylation reactions. Thus, the oxidation state of the iron center modulates not only the rate of HAT but also the rate of ligand rebound

The Two Faces of Tetramethylcyclam in Iron Chemistry: Distinct Fe–O–M Complexes Derived from [Fe^IV(O_{<i>anti</i>/<i>syn</i>})(TMC)]²⁺ Isomers

Tetramethylcyclam (TMC, 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) exhibits two faces in supporting an oxoiron­(IV) moiety, as exemplified by the prototypical [(TMC)­Fe^IV(O_<i>anti</i>)­(NCCH₃)]­(OTf)₂, where <i>anti</i> indicates that the O atom is located on the face opposite all four methyl groups, and the recently reported <i>syn</i> isomer [(TMC)­Fe^IV(O_<i>syn</i>)­(OTf)]­(OTf). The ability to access two isomers of [(TMC)­Fe^IV(O_{<i>anti</i>/<i>syn</i>})] raises the fundamental question of how ligand topology can affect the properties of the metal center. Previously, we have reported the formation of [(CH₃CN)­(TMC)­Fe^III–O_<i>anti</i>–Cr^III(OTf)₄(NCCH₃)] (<b>1</b>) by inner-sphere electron transfer between Cr­(OTf)₂ and [(TMC)­Fe^IV(O_<i>anti</i>)­(NCCH₃)]­(OTf)₂. Herein we demonstrate that a new species <b>2</b> is generated from the reaction between Cr­(OTf)₂ and [(TMC)­Fe^IV(O_<i>syn</i>)­(NCCH₃)]­(OTf)₂, which is formulated as [(TMC)­Fe^III–O_<i>syn</i>–Cr^III(OTf)₄(NCCH₃)] based on its characterization by UV–vis, resonance Raman, Mössbauer, and X-ray absorption spectroscopic methods, as well as electrospray mass spectrometry. Its pre-edge area (30 units) and Fe–O distance (1.77 Å) determined by X-ray absorption spectroscopy are distinctly different from those of <b>1</b> (11-unit pre-edge area and 1.81 Å Fe–O distance) but more closely resemble the values reported for [(TMC)­Fe^III–O_<i>syn</i>–Sc^III(OTf)₄(NCCH₃)] (<b>3</b>, 32-unit pre-edge area and 1.75 Å Fe–O distance). This comparison suggests that <b>2</b> has a square pyramidal iron center like <b>3</b>, rather than the six-coordinate center deduced for <b>1</b>. Density functional theory calculations further validate the structures for <b>1</b> and <b>2</b>. The influence of the distinct TMC topologies on the coordination geometries is further confirmed by the crystal structures of [(Cl)­(TMC)­Fe^III–O_<i>anti</i>–Fe^IIICl₃] (<b>4</b>_<b>Cl</b>) and [(TMC)­Fe^III–O_<i>syn</i>–Fe^IIICl₃]­(OTf) (<b>5</b>). Complexes <b>1</b>–<b>5</b> thus constitute a set of complexes that shed light on ligand topology effects on the coordination chemistry of the oxoiron moiety