Beyond ferryl-mediated hydroxylation: 40 years of the rebound mechanism and C–H activation
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Since our initial report in 1976, the oxygen rebound mechanism has become the consensus mechanistic feature for an expanding variety of enzymatic C–H functionalization reactions and small molecule biomimetic catalysts. For both the biotransformations and models, an initial hydrogen atom abstraction from the substrate (R–H) by high-valent iron-oxo species (Fen=O) generates a substrate radical and a reduced iron hydroxide, [Fen−1–OH ·R]. This caged radical pair then evolves on a complicated energy landscape through a number of reaction pathways, such as oxygen rebound to form R–OH, rebound to a non-oxygen atom affording R–X, electron transfer of the incipient radical to yield a carbocation, R+, desaturation to form olefins, and radical cage escape. These various flavors of the rebound process, often in competition with each other, give rise to the wide range of C–H functionalization reactions performed by iron-containing oxygenases. In this review, we first recount the history of radical rebound mechanisms, their general features, and key intermediates involved. We will discuss in detail the factors that affect the behavior of the initial caged radical pair and the lifetimes of the incipient substrate radicals. Several representative examples of enzymatic C–H transformations are selected to illustrate how the behaviors of the radical pair [Fen−1–OH ·R] determine the eventual reaction outcome. Finally, we discuss the powerful potential of “radical rebound” processes as a general paradigm for developing novel C–H functionalization reactions with synthetic, biomimetic catalysts. We envision that new chemistry will continue to arise by bridging enzymatic “radical rebound” with synthetic organic chemistry.
KeywordsIron Oxygenase C–H activation Rebound Radical Metal oxo
- cpd I
- cpd II
Mechanisms of C–H activation by cytochrome P450s and other iron-containing oxygenases
The heme-thiolate-containing monooxygenases, cytochrome P450, have assumed a uniquely important position in the hierarchy of the field and served as prototypical example to our understanding of the iron-containing oxygenases [22, 23, 24, 25]. P450 enzymes (now termed CYP) catalyze highly selective C–H hydroxylations, as well as epoxidations, desaturations, dealkylations, and C–C bond cleavage reactions in an extremely wide range of compounds. Typical substrates include xenobiotics such as pharmaceuticals and agrochemicals and precursors for the biosynthesis of steroids, terpenoids, alkaloids, antibiotics, pigments, antioxidants, etc. Bacterial P450s have been genetically engineered for large-scale bio-transformations [26, 27].
In spite of these close analogies, the lack of direct spectroscopic and kinetic characterization of a P450 compound I led to proposals of other intermediates such as iron(V)oxo and ferric hydroperoxo as alternative hydrogen-abstracting intermediates for P450s [65, 66, 67]. The long-sought P450 compound I was finally captured in 2010. Using freeze-quench techniques, Rittle and Green successfully obtained the compound I of CYP119 in high yield . The near-zero chemical shift of the iron in the Mössbauer spectrum, the doublet electronic ground state signaled by the EPR spectrum, the weakened and blue-shifted Soret band in the UV, and a long-wavelength absorbance in the visible near 700 nm explicitly showed that CYP119-I is indeed an oxoiron(IV) porphyrin cation radical. These spectral signatures are reminiscent of those found in the first synthetic ferryl porphyrin cation radical (species I, Fig. 5) as well as the reactive [4-TMPyP·+]FeIV=O. CYP119-I was highly reactive toward unactivated C–H bonds with an apparent rate constants in the range of 104–107 M−1 s−1. In 2012, a second reactive compound I was characterized by our group for the extracellular heme-thiolate aromatic peroxygenase from Agrocybe aegerita (AaeAPO) [69, 70]. These newly discovered fungal peroxygenases, now called unspecific peroxygenases (UPO), are only distantly related to chloroperoxidase according to their amino acid sequences, and completely unrelated to CYP enzymes, although the proximal ligand environment, including peptide N–H hydrogen bonding to the heme-thiolate sulfur, is very similar . AaeAPO-I showed fast rate constants for substrate hydroxylations for C–H bonds up to 100 kcal/mol in the range of 10–105 M−1 s−1 [69, 70], confirming compound I as the intermediate for hydrogen atom abstraction of heme-thiolate hydroxylases.
A highly basic compound II (pK a = 10.0, Fig. 7d) was also observed for the heme-thiolate aromatic peroxygenase APO described above . Importantly, the reduction potential of APO-I, 1.2 V with respect to the resting ferric protein, could also be determined through a Nernst equation analysis of its reversible reaction kinetics with chloride and bromide ions. This value allowed for the determination of the individual, one-electron reduction potentials of APO-I and APO-II to be E cpd I/cpd II = 1.4 V and E cpd II/ferric = 0.8 V at pH 7.0, and our estimate that the O–H BDE of Cys–S–FeIV–O–H of APO is ∼100 kcal/mol. What this means is that for APO, and likely P450 as well, strong aliphatic C–H bonds are cleaved by these enzymes rapidly, even though there is little or no driving force for the reaction.
Another example of enzymes that exploit high-valent iron-oxo species for C–H activation are the non-heme diiron hydroxylases. The reactive intermediate for this family of enzymes is a bis-μ-oxo-iron(IV) complex called compound Q [89, 90, 91]. The compound Q of soluble methane monooxygenase (sMMO) has been well characterized [91, 92, 93]. Similar intermediates were also proposed for other non-heme diiron enzymes including the ω-hydroxylase AlkB, toluene monooxygenase (T4moH), and xylene monooxygenase (XylM) [89, 94]. Like compound I in P450s, compound Q initiates a hydrogen abstraction/radical rebound sequence to hydroxylate hydrocarbons, most remarkably, even methane with its 104 kcal/mol C–H bonds (Fig. 8b). From these various enzymatic reactions, it is clear that the radical rebound mechanism is a general paradigm in biological C–H oxidations catalyzed by iron-containing oxygenases, uniting the heme and non-heme enzymes.
The intermediacy of substrate radicals during C–H activation by iron-containing oxygenases
The characteristic feature of the radical rebound mechanism is the intermediacy of the substrate radical generated in the initial hydrogen abstraction step. The properties and behavior of the incipient radical (i.e. lifetimes and conformational changes) and physical and chemical characteristics of the radical rebound step (i.e. rate constant and stereoselectivity) greatly influence the reaction outcome and are of crucial importance for the understanding of chemistries involving high-valent iron-oxo complexes. However, the transient nature of the substrate radical and the radical rebound step involving [Fen−1–OH ·R] has generally precluded direct mechanistic studies with common kinetic and spectroscopic methods. In this regard, mechanistically diagnostic substrates, which form radicals that undergo changes in stereochemistry or structure after hydrogen abstraction, offer a powerful tool to study the intermediate radical and the rebound step .
Typical radical lifetimes of non-heme iron enzymes and synthetic metalloporphyrins determined by norcarane
The large range of radical lifetimes for various enzymes and synthetic model compounds highlights the intricacy and inner diversity of the transient radical rebound step of [Fen−1–OH ·R]. A fundamental question to ask is what factors control the radical lifetimes and rebound rate. From a thermodynamic perspective, radical rebound is a one-electron reduction process to the metal center. It is therefore not surprising that the metal center as well as the oxidation of the substrate radical intermediate would affect the radical rebound rate. This notion is well illustrated by the over tenfold increase in radical lifetimes for manganese porphyrin-catalyzed aliphatic hydroxylations compared to the reactions catalyzed by iron porphyrins (Table 1) [55, 110]. In sharp contrast, radical intermediates were not observed for C–H hydroxylations catalyzed by ruthenium porphyrins [110, 111]. Such variations in rebound behavior likely result from the differences in oxidation potentials, electronic configurations, as well as the relative energetics of different spin states of the rebounding intermediates [111, 112, 113, 114, 115, 116, 117].
In addition to the intrinsic properties of the rebound intermediates, another important, and often overlooked factor that affects the rebound step is the cage effect. After the initial hydrogen atom abstraction, the incipient substrate radical and rebound intermediate comprise a caged radical pair [Fe–OH ·R]. We note that weak interactions between the substrate radical and the iron center at this stage would facilitate spin state interconversions for the ensemble. Another important feature of such caged radical species is the competition between the in-cage radical recombination and the diffusive cage escape. In an enzyme active site a water molecule, or a protein functional group, can insert itself between the substrate radical and the metal center as in [Fe–OH···OH2·R]. There is abundant photophysical evidence for competitive cage-escape and recombination as stochastic events in radical reactions since their initial discovery in 1930s . A compelling example is the homolytic cleavage of carbon-cobalt bond in 5′-deoxyadenosylcobalamin (coenzyme B12) to form cobalt(II)-cobalamin and an adenosyl radical, which is important for the biological functions of coenzyme B12-dependent mutase enzymes. Time-resolved spectroscopic studies showed that the initial radical pair formed after Co–C bond homolysis in adenosylcobalamin undergoes in-cage radical recombination and cage escape both at approximately 109 s−1, clearly indicating a competition between the two processes [124, 125, 126].
The realization of the significant influence of cage effects in radical rebound processes came from our studies of synthetic heme-model compounds. In 1979, we reported the first synthetic iron porphyrin system that effected stereospecific alkane hydroxylation and olefin epoxidation . Further examination of this reaction showed that in the presence of a radical trap, bromotrichloromethane, an 18% yield of the bromination trapping product was obtained . This result clearly indicated the presence of the cage escaped substrate radical that had encountered BrCCl3 in solution.
Very recently, Shaik and Nam have examined the cage escape/radical rebound processes for a number of non-heme metal-oxo systems . They found that cage escape pathways generally showed low calculated energy barriers that could well compete with the in-cage radical recombination processes. In several cases, such as [MnIVO(Bn-TPEN)]2+ and [FeIVO(Bn-TPEN)]]2+ (Bn-TPEN = N-benzyl-N,N′,N′-tris(2-pyridylmethyl)-1,2-ethylenediamine), radical cage escape was found to be the preferred pathway and the diffusing radicals could be trapped by radical scavengers such as CCl3Br, N-bromosuccinamide, or O2 under aerobic conditions [131, 132, 133, 134].
Radical rebound mechanism: a reaction manifold for versatile biotransformations other than oxygenation
Computational studies by de Visser et al. also suggested a slow-down of the radical rebound for OleT . An 8 kcal/mol energy barrier was estimated for the oxygen rebound of substrate radical to OleTJE compound II, while the energy barrier of the decarboxylation pathway was estimated to be below 1 kcal/mol. The calculation also suggested that hydrogen bonding interactions within the P450 OleTJE active site are essential for the destabilization of the oxygen rebound.
Chlorination is not the only reaction that Fe(II)/αKG halogenase could effect. Recently, Bollinger and co-workers found that Fe(II)/αKG halogenases could catalyze C–H azidation and nitration upon replacing the iron-bound chloride with azide and nitrite . Analogous to the chlorine rebound scenario in halogenases, the incipient radical formed after initial C–H abstraction by FeIV=O recombined with an azidoferric (N3–FeIII–OH) or a nitritoferric (NO2–FeIII–OH) intermediate to form the C–N3 or C–NO2 bond, albeit in modest conversions.
An analogous example of HEPD and MPnS is 2-hydroxypropylphosphonate epoxidase (HppE) [166, 167, 168, 169, 170]. HppE is involved in the biosynthesis of the antibiotic fosfomycin from (S)-2-hydroxypropylphosphonate (2S-HPP). In this reaction, an oxoferryl intermediate is formed prior to the epoxide formation step that abstracts the pro-R hydrogen from C1 of 2S-HPP. Subsequent electron transfer from the C1 radical to FeIII–OH and epoxide ring closure afford the fosfomycin product (Fig. 19b).
In addition to the examples discussed herein, there are a number of other reactions that can be effected by iron oxygenases via controlling the reaction pathways of incipient radicals, including stereoinversion, oxacyclization, carbodesaturation, etc. In another variation of the rebound spectrum of reactions, aldehyde deformylation by the diiron enzyme ADO produces alkanes in a process that can be seen as a hydrogen rebound from Fe–OH2 . These reactions have recently been reviewed by Bollinger and Krebs . The common feature of these biotransformations is the involvement of an initial hydrogen atom abstraction by high-valent iron-oxo intermediates. The desired reaction outcome is achieved by directing the incipient substrate radicals to the corresponding radical reaction pathways such as oxygen rebound, non-oxygen atom rebound, electron transfer, radical cage escape, etc. In this way, nature can achieve a diverse array of C–H transformations with molecular oxygen as the oxidant by employing these aspects of the rebound process.
Harnessing the radical rebound mechanism for novel organic radical transformations through biomimetic catalysis
The direct functionalization of aliphatic C–H bonds remains one of the grand challenges for the chemical catalysis community . Activation of aliphatic C–H bonds with organometallic reagents, such as Shilov chemistry or many Pd-catalyzed C–H functionalizations, generally requires harsh conditions or utilizes a directing group approach because of the low acidity and weak coordination capability of aliphatic C–H bonds [174, 175, 176, 177]. On the other hand, it has been long recognized that direct functionalization of aliphatic C–H bonds can be achieved via radical chemistry [178, 179, 180]. A classic example is the radical chlorination of hydrocarbons with Cl2 under photolytic or thermal conditions, which have been used for the industrial synthesis of chloroform and dichloromethane from methane . The power of radical C–H activation has been further demonstrated by the recent renaissance of catalytic radical-based methods, especially photoredox catalysis that has provided a variety of new methods for aliphatic C–H functionalization reactions [182, 183, 184].
There are many catalytic systems for aliphatic C–H hydroxylation reactions based on synthetic models of Fe-containing oxygenases, which have been reviewed extensively [17, 18, 185]. Herein, we will mainly focus on nonoxygenation reactions via the radical rebound mechanism, which has offered solutions to several most challenging reactions in synthetic organic chemistry.
Radical clock studies showed that the reaction followed a radical mechanism with a radical lifetime ~11 ns, which is much longer than that of the analogous hydroxylation (~2 ns). No cation rearranged product was observed, which further indicated a radical rebound mechanism.
This series of unprecedented radical C–H functionalization reactions share common features and mechanistic foundations. They are all initiated by hydrogen abstraction via oxoMnV intermediates, which give rise to the tunable regioselectivity that can surpass the restriction of innate reactivity of aliphatic C–H bonds. The oxygen rebound in these reactions are all suppressed due to the presence of strong anionic σ-donating ligands. Although more studies are needed to further elucidate the nature of non-oxygen rebounding intermediate, radical clock studies with norcarane showed long radical lifetimes (~2 ns for fluorination, ~10 ns for chlorination, and ~30 ns for azidation). The long radical lifetime seems to be crucial for the success of non-oxygenation reactivity, as changing the catalyst to more electron-withdrawing manganese porphyrins [such as Mn(TPFPP)Cl or Mn(TDCPP)Cl] or iron porphyrins suppresses the non-oxygenation reactivity and yields mainly oxygenation products. The stereoselectivity and the enantioselectivity of the reactions can be controlled by the catalyst through metal-mediated radical rebound. Such control of stereoselectivity is difficult to achieve with simple organic or inorganic radical trapping reagents.
The above examples show just a glimpse of the potential applications of radical rebound strategy in developing new radical organic transformations. Both the C–H activation and radical recombination approaches adopted by iron-containing oxygenases could likely be combined with current catalytic radical methodologies to afford new radical transformations. In one scenario, one can imagine that the carbon-centered radicals can be generated by the C–H activation via metal-oxo or similar metal-based hydrogen abstracting intermediates and subsequently be redirected to carbon–carbon or carbon-heteroatom bond formation via transition-metal mediated transformations. Such reactions would harness the controllable selectivity of metal-based hydrogen-abstracting intermediate in C–H activation step, which is hard to achieve with simple organic or inorganic radicals. In another case, carbon radicals could be generated with other radical initiation methods such as photoredox reactions, electrolysis, or other single-electron transfer (SET) processes, and later be captured by metal complexes [213, 214, 215]. The works of Kochi in 1970s have provided early examples of this type of reactions. He showed that various metal halide or pseudohalide complexes [i.e. CuBr2, Pb(OAc)4, etc.] were able to trap alkyl radicals generated by photolysis or thermolysis to afford halogenation or pseudohalogenation products [210, 211, 216]. Very recently, this area of research has seen dramatic progress mainly due to the development of photoredox catalysis. New methodologies have been developed for the construction of various carbon–carbon and carbon-heteroatom bonds via trapping the intermediate carbon radicals with transition-metal complexes especially nickel and copper [214, 217, 218, 219, 220, 221, 222, 223].
In addition to non-oxygen atom rebound, other reaction pathways adopted by iron-containing oxygenases are also highly valuable to the organic synthesis, such as alkane desaturation and radical cyclizations. There are relatively few studies on these reactions. The elucidation of their biochemical mechanisms and the design of synthetic model compounds to mimic the reactivities would definitely lead to new inventions in organic synthesis.
It has been 40 years since our initial work on the radical rebound mechanism. The goals all along were to compare, contrast, and unify biocatalytic mechanisms with biomimetic systems. Iron-containing oxygenases continue to comprise a highly active research area with numerous new enzymes and numerous new reaction discoveries being made every year. What is fascinating about these enzymes is the diverse range of transformations they can catalyze. What is even more intriguing is the common mechanistic foundation underlying these various reactions: activation of oxygen to form high-valent iron–oxygen containing intermediates; abstraction of C–H bonds to yield radical intermediate; and the participation of the carbon-centered radicals in different reaction pathways giving rise to the diverse reactivities observed for iron-containing oxygenases. The study of the fundamental reactivities of these enzymes has inspired critical breakthroughs in synthetic organic chemistry, from early development of new molecular catalysts for alkane hydroxylations to the most recent progress in C–H halogenation reactions. We can expect iron-containing enzymes to continue powering the discoveries of new organic transformations because of the continuous discoveries and understanding of new reactivities of these enzymes as well as the recent renaissance of radical chemistry in organic synthesis.
Deepest thanks are due to all the co-workers from the Groves laboratories, both at the University of Michigan, for the earliest work, and Princeton University. JTG thanks Ronald Breslow for stimulating an early interest in biomimetic chemistry, Cheves Walling for deep insights regarding kinetics and mechanism and Jud Coon and Vince Massey for an introduction to oxygenases. We thank those involved in the metalloporphyrin and P450/APO studies referred to in this review for their invaluable intellectual and experimental contributions. We are especially indebted to Dr. Wei Liu for his contributions to the discovery and development of the Mn–F chemistry. Mn-porphyrin research was supported by the U.S. National Science Foundation Awards CHE-1148597 and CHE-1464578 and the Center for Catalytic Hydrocarbon Functionalization, an Energy Frontier Research Center, U.S. Department of Energy, Office of Science, Basic Energy Sciences, under Award No. DESC0001298. P450 and APO enzymology was supported by the National Institutes of Health (2R37 GM036298). XYH thanks Merck, Inc. for fellowship support.
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