Homogeneous Oxygenase Catalysis - Chemical Reviews (ACS

Review Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

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Homogeneous Oxygenase Catalysis Yujie Liang,† Jialiang Wei,† Xu Qiu,† and Ning Jiao*,†,‡ †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan Road 38, Beijing 100191, China ‡ State Key Laboratory of Organometallic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China ABSTRACT: Oxygenases-catalyzed reductive activation of molecular oxygen and incorporation of O atoms into an organic molecule is undoubtedly one of the most attractive research areas. Typically, these oxygenation reactions proceed with high selectivity and reactivity, which is seldom found in its “biomimetic” chemocatalytic counterparts. Furthermore, enzymatic oxygenation can avoid undesired overoxidation, which is frequently observed in (industrial) chemical transformation. Therefore, it is not surprising that tremendous attention has been paid to enzymatic oxygenation. Their application in organic synthesis has been steadily growing over the years. The goal of the present Review is to provide a handy reference for chemists interested in using homogeneous oxygenase catalysis and those interested in discovering new types of biomimetic oxidations and oxygenations with dioxygen. In this Review, we will review the recent advances in in homogeneous oxygenase catalysis to reveal the great achievements and potentials in this field.

CONTENTS 1. Introduction 1.1. Background 1.2. Flavin-Dependent Monooxygenases 1.3. Heme-Dependent Monooxygenases 2. Homogeneous Oxygenase-Catalyzed Oxygenations 2.1. Enzymatic Baeyer−Villiger Oxidation 2.1.1. Oxidation of Ketones and Aldehydes 2.2. Oxidation of Sulfides 2.2.1. Enantioselective Sulfoxidations 2.3. Oxidation of Amines, Boranes, and Selenium Compounds 2.4. Hydroxylation of Arenes 2.4.1. Using Flavin-Dependent Monooxygenases as Catalyst 2.4.2. Using P450 Monooxygenases as Catalyst 2.4.3. Using Tyrosinase As Catalyst 2.5. Oxygenation of CC Double Bonds 2.5.1. Epoxidation of CC Double Bonds 2.5.2. Oxidative Cleavage of CC Double Bonds 2.6. Oxygenation of sp3 C−H Bonds 2.6.1. Selective Hydroxylation of Structurally Simple Molecules 2.6.2. Selective Hydroxylation of Complex Molecules 3. Summary and Outlook Associated Content Special Issue Paper Author Information Corresponding Author © XXXX American Chemical Society

ORCID Notes Biographies Acknowledgments Abbreviations References

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1. INTRODUCTION 1.1. Background

Biocatalytic reactions generally operate under mild reaction conditions and exert remarkable regio-, chemo-, and enantioselectivity, particularly for structurally complex molecules that cannot tolerate harsh reaction conditions.1−4 Among them, oxygenases-catalyzed reductive activation of molecular oxygen and incorporation of O atoms into an organic molecule is undoubtedly one of the most attractive research areas.5,6 These oxygenation reactions proceed with high selectivity and reactivity that is seldom found in its “biomimetic” chemocatalytic counterparts.7 It is generally believed that the high selectivity and reactivity stems from the unique embedment of a highly reactive oxyferryl complex or organocatalysts in the cavity of an enzyme’s active site. The well-defined 3D framework of the protein positions the starting substrates precisely toward the active catalytically species (accounting for selectivity) and stabilizes the transition states, thereby leading to dramatic acceleration of reaction rates.8,9 Furthermore, enzymatic oxygenation can avoid undesired overoxidation, which is frequently observed in (industrial) chemical trans-

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Received: April 7, 2017

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DOI: 10.1021/acs.chemrev.7b00193 Chem. Rev. XXXX, XXX, XXX−XXX

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formation.8 Therefore, it is not surprising that tremendous attention has been paid in the enzymatic oxygenation, and its application in organic synthesis has been steadily growing over the years. The goal of this Review is to provide a handy reference for chemists interested in using homogeneous oxygenase catalysis, as well as those interested in discovering new types of biomimetic oxidations and oxygenations with dioxygen. In this Review, we will discuss the recent advances in homogeneous oxygenases catalysis to reveal the great achievements and potentials in this field. To meet the objective of this Review, the scope is limited to catalytic systems involving direct interaction of enzymatic active sites with molecular oxygen. Enzymes that employ reduced O2 derivatives such as H2O2 as a substrate will thus be excluded. In particular, the most common oxygenases, including flavindependent monooxygenases and heme-dependent oxygenases such as P450-monooxygenases, will be covered. Other substrate-specific monooxygenases such as tyrosinase will be highlighted with mechanistic emphasis. Dioxygenases as a heterogeneous group of enzymes that catalyze the introduction of both oxygen atoms to the substrates10 will not be emphasized in this Review. It is noteworthy that nonheme iron-dependent oxygenases can also facilitate the incorporation of oxygen into biomolecules;11−15 the demonstration of the enzyme models was reviewed by Professor Que16 in the “Oxygen Reduction and Activation in Catalysis” thematic issue of Chemical Reviews. Moreover, catalytic properties and synthetic contexts of oxygenases or oxidases with mononuclear Mn, Co, and Ni active sites have also been beautifully summarized.17 Considering some very nice reviews have summarized the catalytic mechanisms, structure, and function of the enzymes,6,13,14,17−22 this Review will focus on demonstrating reactions. Throughout this Review, regioselective oxygenation of nonactivated C−H and CC bonds and the oxidation of ketones, aldehydes, and carboxylic acids as well as the heteroatom oxygenations will be carefully introduced. Also highlighted is recent progress made in the areas of new enzyme development, especially the existing ones that have been improved to meet the requirement of synthetic applications. An effort was made to be comprehensive; however, taking into account the recent reviews highlighting oxygenase-involving reactions, in this contribution, the focus is on presenting latest advances and findings on synthetic interest.

Scheme 1. C4a-(Hydro)peroxoflavin as the Catalytically Active Species Catalyzed Diverse Oxygenation by FlavinDependent Monooxygenases

reactions are flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD). Flavin cofactors have to be reduced in order for the electron-rich species to react with molecular oxygen as a substrate; it is a catalytic mechanism and therefore contains a reductive and an oxidative half-reaction. It is generally believed that the reaction initiates the NAD(P)Hmediated reduction of the enzyme-bound flavin group A. From this, the reduced flavin intermediate B then reacts with O2 to afford an organic peroxide intermediate. In most cases, a covalent adduct between C4a of the flavin and O2 is generated and stabilized, yielding a reactive C4a-hydroperoxyflavin species C. This catalytically active species C subsequently performs the oxygenation through nucleophilic or electrophilic attack on the substrates. Finally, the resting oxidized state is regenerated after extrusion of water of D (Scheme 2).23 It should be noted that the understanding of the detailed mechanism of how flavinScheme 2. Simplified Catalytic Cycle of Flavin-Dependent Monooxygenases (Protonation Steps Omitted for Simplicity)

1.2. Flavin-Dependent Monooxygenases

The direct interaction between ground-state O2 (triplet) with organic molecules (singlet) is spin-forbidden; therefore, the enzyme has to be capable of activating molecular oxygen. Various enzymes have found their way to employ molecular oxygen as a substrate to oxygenate organic compounds. Flavindependent monooxygenases are a group of enzymes capable of catalyzing various synthetically useful oxygenation reactions, including aromatic C−H hydroxylation, cleavage of C−C bonds, epoxidation of CC double bonds, and enantioselective heteroatoms oxygenation (Scheme 1).23−28 They incorporate one O atom of dioxygen into their target substrates, while the second atom is reduced to water. All oxygenases require a cofactor to activate O2. They utilize a purely organic cofactor to perform oxygenation in a wide set of substrates. The most common flavin cofactors that are harbored by flavoenzymes to participate in various oxygenation B


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better understanding the heme-iron-dependent enzyme-catalyzed systems, including the role of thiolate ligation in facilitation of C−H bonds activation.35 The oxygen rebound mechanism has become the consensus catalytic feature of various enzymatic C−H activation for the past few decades and has also been applied for versatile biotransformations other than oxygenation.36 Monooxygenases are NAD(P)H-dependent, whereas wholecell biocatalysis affords the required enzymes and cofactors. However, if purified enzymes are employed to catalyze the reaction, nicotinamide cofactors need to be provided externally. In general, the reducing equivalents required for reductive activation of molecular oxygen are obtained from NAD(P)H, which provides two electrons to the P450-monooxygenases indirectly via a reductase/mediator cascade. Because of the high costs of NAD(P)H, an efficient cofactor recycling system is required so that the NAD(P)H can be applied in catalytic amounts. Some reviews have significantly summarized the detailed discussion of the NAD(P)H regeneration strategies.8,9,37,38 Available wild-type (WT) CYPs are not routinely successful in the oxygenation of organic compounds. Protein engineering has emerged as a powerful tool in remodelling P450s in the oxygenation of non-native substrates by enhancing both reactivity and selectivity. Indeed, engineered P450s have displayed incredible potential in the direct oxygenation of a diverse range of organic molecules.21,39,40 Next to protein engineering, substrate engineering is another effective method to enhance P450 catalysis by utilizing small molecules to control the substrate specificity and product selectivity of P450s, focusing on either taking advantage of noncovalent decoy molecules or covalent substrate modifications.41−43 It is worth mentioning that, in contrast to P450monooxygenases that catalyze the reductive activation of molecular oxygen, peroxygenases can use the hydrogen peroxide directly to generate the catalytically active oxyferryl species via a so-called hydrogen peroxide shunt pathway to perform the O-transfer reaction.44−46 Recently, a new generation of X-ray free-electron lasers that possess the ability to generate intense X-rays on the femtosecond time scale have been utilized to carry out structure determination of cytochrome c peroxidase compound I. This is done with no X-ray damage or reduction of metal centers, thus aiding the understanding of the catalytic mechanism, not only for peroxidases but also for cytochrome P450.47

dependent monooxygenases activate oxygen is still one of the most challenging tasks in flavoenzymology.29 Additionally, by flavin cofactor redesign, flavoproteins can be engineered to function as peroxygenases, allowing for H2O2-driven oxygenation.24,30 Recently, a rare case revealed that the reducing equivalents can come from the substrate itself in the bacterial flavoenzyme EncM-catalyzed biosynthesis of the antibiotic enterocin.31 1.3. Heme-Dependent Monooxygenases

Heme-dependent monooxygenases, also called CYP- or P450monooxygenases, are one of the most well-known monooxygenases and are ubiquitously distributed enzymes that perform the biooxidation of steroids, eicosanoids, and fatty acids. These heme-containing enzymes are known to be capable of oxygenating C−H bonds. The heme cofactor of these enzymes contains a protoporphyrin IX-bound iron atom; this atom is bound to an active site residue, leaving a single coordinating site accessible for ligand binding and catalysis. These can react with molecular oxygen to generate catalytically active species to enable oxygenation of a wide set of substrates. Furthermore, they play important roles in protecting organisms from toxins and xenobiotics. Numerous enzymes belonging to the P450 family are therefore revealed and characterized. They can abstract nonactivated C−H bonds and oxygenate a diverse range of substrates. The reactions mainly include hydroxylation of alkane and aromatic rings, epoxidation of CC double bonds, and heteroatom oxygenations (Scheme 3). Their catalytic properties have been extensively studied over the past several decades.18−21,32−34 Scheme 3. Representative Examples of P450 Monooxygenases-Catalyzed Oxygenation

2. HOMOGENEOUS OXYGENASE-CATALYZED OXYGENATIONS

The mechanism of P450-monooxygenase-catalyzed oxygenation is somewhat more complicated than flavin-dependent monooxygenases. The catalytic cycle begins with binding of the substrate to the resting state Fe(III) A in the active site of the enzyme to form intermediate B. This is followed by the single electron transfer from NAD(P)H to furnish intermediate C. Subsequently, the oxygen binding of intermediate C affords intermediate D. This then undergoes the second electron transfer to generate a ferric peroxide adduct E. The intermediate E can be protonated to yield a hydroperoxide iron complex F, which is dehydrated after successive protonation, and a highly oxidized oxoferryl porphyrin cation radical intermediate G is produced. H atom abstraction of the substrate by G and the following radical recombination form the new C−O bonds (Scheme 4).32−34 Many constructive mechanistic studies and discussions have been devoted to

2.1. Enzymatic Baeyer−Villiger Oxidation

Baeyer−Villiger oxidation is a key synthetic transformation that proceeds with regioselective C−C bond cleavage and oxygen insertion. However, these chemical strategies suffer from several limitations: they often lack specificity and require toxic and explosive chemical reagents. Baeyer−Villiger monooxygenases (BVMOs) represent a group of oxidative enzymes that are capable of performing Baeyer−Villiger-type oxidations generally with high chemo-, regio-, and/or enantioselectivity under mild reaction conditions.25,26 All known BVMOs characterized so far are NAD(P)H-dependent, flavin-containing enzymes. Due to their importance in organic synthesis, the substrate spectrum of enzymatic Baeyer−Villiger oxidations has been C


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Scheme 4. Very Simplified Catalytic Cycle of P450 Monooxygenases

significantly expanded, and lots of useful reactions have been developed in the past several decades by synthetic chemists.25,26 The most extensively studied BVMO is cyclohexanone monooxygenase from Acinetobacter calcoaceyicus NCIB 9871 (CHMOAcineto), which displays an exceptionally broad acceptance of substrates. In fact, this enzyme has been employed in hundreds of oxidation procedures. Various ketones, including linear, cyclic, bicyclic, and heterocyclic with varied ring sizes and different substitution patterns, can be easily oxygenated. Moreover, sulfides and dithienes can also be oxidized to the corresponding chiral sulfoxides. It should be outlined that CHMOAcineto has been used as a readily accessible synthetic tool in organic synthesis through protein engineering.48 In addition to cyclohexanone monooxygenases, phenylacetone monooxygenase (PAMO) from the thermophilic bacterium Thermobif ida f usca is another promising BVMO that is worthy of special attention. This is a particularly interesting BVMO because it is thermostable and tolerates various organic solvents. Due to the stability of this enzyme, its crystal structure has been obtained, and its catalytic mechanism has been elucidated. The first crystal structure of phenylacetone monooxygenases not only provides deep insight into the mechanism of enzymatic Baeyer−Villiger oxidations but also enables structure-guided protein engineering to broaden the substrates scope.49 Phenylacetone monooxygenases mostly accept aromatic ketones such as phenylacetone and 4-hydroxyacetophenone; however, they are not able to oxidize cyclohexanone.48 Comprehensive reviews on BVMOs catalysis, including their application in organic synthesis and the elucidation of their crystal structure as well as mechanistic studies, are available.48−51 Therefore, this part will only give an overview of representative synthetic applications of BVMOs and present some of the latest interesting findings. 2.1.1. Oxidation of Ketones and Aldehydes. BVMOs catalyze the oxidation of ketones and aldehydes, and these reactions are a hot topic in enzymatic biocatalysis. Cyclic, linear, and aromatic ketones can be oxidized to the respective lactones and esters. For aldehydes 1, two different products can be yielded through Baeyer−Villiger oxidation, namely, the corresponding acid 2 and ester (or formate) 3. The selectivity of these two products depends on the different steric hindrances and electronic natures of the substrates. Recently, BVMO4, a new BVMO from Dietzia sp. D5, was shown to be

effective in the oxidation of several aromatic and aliphatic aldehydes. Interestingly, this enzyme catalyzed the preferential formation of the corresponding carboxylic acid. Complete regioisomeric excess (RE) was observed for the oxidation of aliphatic aldehydes such as octanal and decanal (Scheme 5).52 Scheme 5. Oxidation of Aldehyde Substrates with BVMO4

The mechanism of BVMOs CHMOAcineto catalyzed oxidation of cyclohexanone has been comprehensively investigated. To begin with, the FAD molecule A is reduced by NADPH to generate the intermediate B, which then reacts with O2 to form a C4a-peroxyflavin intermediate C. Intermediate C then undergoes nucleophilic attack of the ketone substrate, producing the “Criegee adduct” D that subsequently rearranges to afford the C4a-hydroxyflavin E and the lactone. The oxidized FAD is regenerated after dehydration of the C4a-hydroxyflavin E. (Scheme 6).53 Some recent representative examples of oxygenation reactions mediated by BVMOs are presented here. Opperman and co-workers reported the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. This is the first fungal BVMO that converts linear ketones with high regioselectivity, representing the highest rates obtained to date for BVMO-catalyzed transformations of 2alkanones. It showed the best conversion of alkanones 4 with chain lengths from C8 to C12 (Scheme 7).54 It should be noted that some BVMOs hold great potential for industrial synthesis of bulk chemicals. For example, recently, Fink and Mihovilovic reported that 3-hydroxypropionates can be successfully synthesized starting from levulinic acid derivatives. The desired products can be produced at ambient temperature in water using O2 as oxidant. These compounds D


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Scheme 6. Mechanism of CHMOAcineto Catalyzed Oxidation of Cyclohexanone

Scheme 7. BVMOAFL838 Catalyzed Oxygenation of Alkanones

Scheme 8. BVMO-Catalyzed Biotransformation of Ricinoleic Acid 5 into Ester 6

can be readily hydrolyzed to 3-hydroxypropionic acid, a versatile precursor of formalonates, acrylates, and 1,3-propanediol.55 More recently, Park and co-workers investigated engineering of the BVMO from P. putida KT2440 and the gene expression system to enhance its catalytic activity and stability for large-scale conversion of ricinoleic acid 5 to ester 6. The polyionic tag-based BVMO engineering and the application of synthetic promoter-driven constitutive gene expression allowed the E. coli-based whole-cell system to produce ester 6 to 85 mM (26.6 g/L) within 5 h. Moreover, the reaction can be scaled to 70 L. This work will contribute to industrial applications of a BVMO-based whole-cell system (Scheme 8).56 Methyl propanoate is of industrial interest as a precursor to acrylic plastic; the development of efficient synthetic methods for the access of methyl propanoate is very attractive. Good selectivity for the abnormal product was achieved by Fraaije and co-workers by using CHMOAcineto catalyzed oxidation of 2butanone 7.57 To enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate 8, various residues near the substrate and NADP+ binding sites in CHMOAcineto were subjected to saturation mutagenesis. The T56S/I491A CHMOAcineto mutant exhibits a significant improvement in both 2-butanone 7 conversion (73%) and the regioselectivity toward methyl propanoate 8 (43%) as compared to those of WT CHMOAcineto. This chemistry shows that, even for a relatively small aliphatic substrate such as 2-

butanone 7, catalytic efficiency and regioselectivity can be tuned by structure-inspired enzyme engineering (Scheme 9).58 Scheme 9. BVMO-Catalyzed Oxidation of 2-Butanone into the Methyl Propanoate

Tang and co-workers discovered that FR9H-Ox, an FADdependent BVMO-tailoring domain, can catalyze the Baeyer− Villiger oxidation of ACP-tethered thioester 9 to form an ACPlinked thiocarbonate 10 (ACP is acyl carrier protein). Further investigation revealed that the interaction between FR9H-Ox and the ACP domain is crucial for FR9H-Ox to perform this BV oxidation (Scheme 10).59 Biocatalytic synthesis of optically pure lactone is important because many of them are essential intermediates of some natural products and bioactive compounds.60 A nice recent example revealed that CHMO-type BVMOs perform best with the oxidation of nitriloketones for the synthesis of chiral nitrilolactones (good yields and excellent ee were obtained), E


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gives the opposite Z-lactones 17. It should be noted that, when using traditional Baeyer−Villiger reagent meta-chloroperoxybenzoic acid (m-CPBA) as the oxidant, only 1:1 mixtures of E-and Z-olefins are produced. Therefore, the biocatalytic approaches displayed superior selectivity (Table 1).63 BVMOs have also played key roles in the oxidation of complex molecules and natural products.28,64 Recently, in the biocatalytic synthesis of pentalenolactone, the group of Cane and Zhu reported that PenE and PntE, orthologous proteins from Streptomyces exfoliatus and S. arenae, respectively, can catalyze the BV oxidation of 1-deoxy-11-oxopentalenic acid 18 to the lactone pentalenolactone D 19, resulting from the migration of less-substituted methylene carbon. In contrast, the paralogous PtlE enzyme from S. avermitilis catalyzes the oxidation of 18 to neopentalenolactone D 20, in which the more-substituted methane substitution has migrated (Scheme 13). Besides, the key amino acids that affect the regiospecificity of these two classes of BVMOs have been identified by analysis of 13 single and multiple mutants of PntE. The L185S mutation in PntE reversed the observed regiospecificity of PntE; thus, all recombinant PntE mutants harboring this L185S mutation acquired the characteristic regiospecificity of PtlE, catalyzing the conversion of 18 to 20 as the major product (Scheme 14).65 Very recently, a novel BVMO from the thermophilic fungus Thermothelomyces thermophile was reported by Fraaije, Mattevi, and co-workers. This fungal enzyme displays excellent enantioselectivity, acts on various ketones (linear, cyclic, substituted cyclic, and bicyclic ketones), and is particularly active on polycyclic molecules such as steroids (Figure 1), converting them with high efficiency; it is therefore named polycyclic ketone monooxygenase (PockeMO). Extensive studies implicated that this enzyme is thermostable. Moreover, the structure of this enzyme has been determined, laying the basis for further enzyme engineering (Figure 2).66 The transformation of a ketone into a carbonate in natural product biosynthesis is unprecedented. A rare example reported by Tang and co-workers showed that CcsB, a multifunctional BVMO, can catalyze the formation of an in-line carbonate in the macrocyclic portion of cytochalasin E 21. They identified the enzymatic basis for forming the in-line carbonate moiety in cytochalasins isolated from fungi, providing a synthetically useful strategy for conversion of ketones to carbonates (Scheme 15).67 It is worth mentioning that low stability of many BVMOs to a large extent hindered their application in industry. Remarkably, Fraaije and co-workers discovered a robust CHMO from Thermocrispum municipal (TmCHMO) that holds great potential as an oxidative biocatalyst. The enzyme can efficiently convert a range of aliphatic, aromatic, and cyclic ketones. It is intriguing to find that this enzyme is much more thermostable and solvent-tolerant than known CHMOs. Moreover, its crystal structure has been elucidated and an effective recombinant production system has also been established (Figure 3). The thermostability and the exceptional solvent tolerance make it very attractive for biotechnology.68

Scheme 10. FR9H-Ox-Catalyzed Oxidation of ACP-Tethered Thioester To Form an ACP-Linked Thiocarbonate

with CPMO-types serving as a useful extension for different selectivities (Scheme 11).61 Scheme 11. BVMO-Catalyzed Oxidation of Nitriloketones for the Synthesis of Chiral Nitrilolactones

Conjugated ene-lactones and enol-lactones are valuable intermediates in organic synthesis. BVMOOcean, from Oceanicola batsensis DSM 15984, and BVMOParvi, from Parvibaculum lavamentivorans DSM 13023, were selected as the biocatalysts for the synthesis of such lactones. In the reaction with E. coli BL21(DE3) containing BVMOOcean, oxygen insertion occurred between the carbonyl group and the nonethylenic carbon atom of 11 to furnish conjugated ene-lactones 12 (Scheme 12a). Scheme 12. BVMO-Catalyzed Synthesis of Conjugated Enelactones and Enol-lactones

Enol-lactone products 14 were exclusively produced with E. coli BL21(DE3) containing BVMOParvi, resulting from the oxygen atom insertion between the carbonyl group and the double bond of 13 (Scheme 12b).62 Using BVMOs as catalysts, diastereoselective desymmetrization of 4-ethylidenecyclohexanones 15 could afford the corresponding E- or Z-configurated lactones in high selectivity. Utilizing WT CHMOAcineto as a catalyst delivers the E-isomer 16 as the dominant product, while a directed evolution mutant Phe432Ile/Thr433Gly/Leu143Met/Phe505Cys (variant III)

2.2. Oxidation of Sulfides

2.2.1. Enantioselective Sulfoxidations. Chiral sulfoxides are valuable building blocks in organic synthesis, and their synthetic application has attracted tremendous attention. They have been widely utilized as chiral auxiliaries and intermediates in asymmetric synthesis. The high asymmetric induction can be F


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Table 1. BVMO-Catalyzed Diastereoselective Desymmetrization of 4-Ethylidenecyclohexanones

ketone

product

catalyst

E/Z

conversion (%)

time (h)

other products (%)

15a 15b 15c 15d 15b 15d

16a 16b 16c 16d 17b 17d

WT CHMOAcineto WT CHMOAcineto WT CHMOAcineto WT CHMOAcineto variant III variant III

98/2 99/1 >99/1 96/4 20/80 4/96

>99 >99 39 42 30 98

5 5 12 12 12 12

1 4 84 70 7

Scheme 13. BVMO-Catalyzed Oxidation of 18 into Lactones 19 and 20

Scheme 14. Proposed Stereoelectronic Model for the BVMO-Catalyzed Oxidation of 18 into Lactones 19 and 20

Figure 1. PockeMO-catalyzed regioselective oxidation of steroids.

These reactions display remarkable chemoselectivity and functional group tolerance, and “overoxidation products” are usually not observed. Mechanistic studies revealed that, in the oxidation of ketones, the deprotonated C4a-peroxoanion acts as the nucleophile to attack the carbonyl group to form the corresponding “Criegee adduct”. However, in the heteroatom oxidation, the heteroatom such as sulfur serves as the nucleophile to attack the terminal oxygen of the catalytically active species of C4a-hydroperoxyflavin species (Figure 4).51 Several frequently used monooxygenases in the enantiospecific sulfoxidations are presented here. Cyclohexanone monooxygenase CHMOAcineto is a well-studied catalyst capable of oxidizing various sulfur-containing compounds. The application

achieved by the chiral sulfinyl fragment, and the induced optical activities are very versatile. Because of their importance, significant effort has been made in the development of efficient methods for their preparation.69 There are three main routes to prepare the optically active sulfoxides: (1) nucleophilic substitution of chiral sulfur precursor; (2) asymmetric sulfoxidation of prochiral sulfides; (3) kinetic resolution of racemic sulfoxides. Biocatalysis without doubt represents a mild and environmentally friendly way to synthesize these optically active sulfoxides. 2.2.1.1. Using Baeyer−Villiger Monooxygenases. Generally, the key biocatalysts required for these processes are flavindependent enzymes.70 BVMOs are capable of heteroatoms oxygenation, particularly in the enantiospecific sulfoxidation. G


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Figure 4. Ambivalent reactivity of C4a-hydroperoxyflavin in the BV and sulfide oxidations.

BVMO, displaying extensive application in biocatalysis. This enzyme, when coupled with G6P/G6PDH, was capable of oxidizing methyl phenyl sulfides 22 to (S)-methyl phenyl sulfoxides 23 in excellent conversion and enantioselectivity. Reactions of other alkyl phenyl sulfides with longer alkyl chains also proceeded smoothly with HAPMO (Scheme 16).72 Scheme 16. HAPMO-Catalyzed Oxidization of Alkyl Phenyl Sulfides

Figure 2. Overall structure of PockeMO (PDB code 5MQ6). The Nterminal extension (residues 1−73; dark red) is specific to this BVMO enzyme subclass. FAD is yellow, and NADP+ is blue (nicotinamide ring is disordered).

Scheme 15. CcsB-Catalyzed Transformation of Ketones into Carbonates

of this enzyme in the preparation of asymmetric sulfoxides has been significantly demonstrated.51 Phenylacetone monooxygenase (PAMO) is another promising catalyst for enantioselective sulfoxidations. Due to its good thermostability and solvent tolerance, it is attractive for practical applications. However, PAMO has a somewhat narrow substrate scope favoring relatively simple aromatic compounds. Furthermore, PAMO was not suitable for the synthesis of alkyl heteroaryl sulfoxides because generally low activities and/or selectivities were obtained. In addition to aromatic substrates, PAMO can also convert nonaromatic substrates, albeit in moderate conversion and enantioselectivity.71 4-Hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas f luorescens ACB is another frequently used

Moreover, HAPMO also has been shown to be effective for the preparation of alkyl heteroaryl sulfoxides. For nonaromatic sulfides, HAPMO also displayed good reactivity and selectivity (Scheme 17).71 Additionally, both HAPMO and PAMO can perform the desymmetrization of prochiral sulfides 24 and catalyze the

Figure 3. TmCHMO: reduced enzyme bound to NADP+ (left, PDB code 5M0Z); crystal structure of TmCHMO in the oxidized state with a bound nicotinamide (right, PDB code 5M10). H


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(Scheme 20). Moreover, this enzyme has been utilized for the manufacture of esomeprazole in a 100 kg scale, representing the only industrial example of a BVMO so far.75,76

Scheme 17. HAPMO-Catalyzed Oxidization of Sulfides

Scheme 20. BVMO-Mediated Enzymatic Process for Esomeprazole Production

2.2.1.2. Using Other Enzymes. It should be noted that some styrene monooxygenases (SMOs) can also perform sulfoxidations.77 E. coli BL21 (DE3) cells expressing SMO from P. putida CA-3 have been used for enantioselective sulfoxidations. The WT enzyme and an engineered SMO (SMOeng R3-11) can catalyze the sulfoxidation of some thioanisoles, albeit in low to moderate selectivities. These two SMO enzymes can also catalyze the oxidation of benzo[b]thiophene, but the final sulfoxides undergo racemization quickly under the reaction conditions.78 Cyptochrome P450-monooxygenases are also capable of performing enantioselective sulfoxidations, but this activity is hardly exploited for synthetic applications. It was reported that the self-sufficient bacterial P450-monooxygenase (P450SMO) from Rhodococcus sp. ECU0066 can convert some aryl aliphatic sulfides 31 to the corresponding (S)-sulfoxides 32 with good to high enantiomeric excess (Scheme 21).79 The expression of

kinetic resolution of racemic sulfoxides by oxidizing selectively one of the sulfoxide enantiomers to sulfone 25, leaving the other sulfoxide 26 enantioenriched (Scheme 18).71,73 Scheme 18. HAPMO- and PAMO-Catalyzed Kinetic Resolution of Racemic Sulfoxides

Scheme 21. P450SMO Catalyzed Oxidation of Aryl Aliphatic Sulfides

Notably, structure-based directed evolution has been applied to PAMO to obtain a mutant that exhibits reversed enantioselectivity in the asymmetric sulfoxidation of prochiral thioethers. For oxidation of p-methylbenzyl methyl thioether 27, using WT PAMO as a catalyst leads to 90% ee of (S)sulfoxide 28; when using the evolved mutant I67Q/P440F/ A442N/L443I by knowledge-driven iterative saturation mutagenesis (ISM), (R)-selective sulfoxide 29 with 95% ee was obtained (Scheme 19).74 Significantly, BVMOs have also found applications in pharmaceutical chemistry. The Nexium (esomeprazole) oxidation that utilizes engineered BVMOs for chiral sulfoxide production is a nice example. WT CHMO from Acinetobacter sp. was selected as the starting BVMO for esomeprazole oxidation; however, the volumetric productivity of WT CHMO was very poor. Evolution had to be conducted for an optimized process capable of producing the desired product with high yield as well as high ee. Subsequently, after completion of 20 rounds of evolution, the evolved CHMO was capable of producing the desired (S)-enantiomer 30 with up to 99.8% ee

P450SMO was further optimized, leading to significantly enhanced conversion of sulfides and products enantiomeric excess.80 To avoid the constant supply of NAD(P)H of the above catalytic system, recombinant E. coli BL21 (pET28aP450-GDH) whole cell is employed as a biocatalyst.81 Cytochrome P450RhF (CYP116B2) from Rhodococcus sp. NCIMB 9784 was also used for enantioselective sulfoxidations. The reactions proceeded with high substrate conversion and stereoselectivity.82 Recently, a new member of the CYP116B

Scheme 19. PAMO-Catalyzed Oxidation of p-Methylbenzyl Methyl Thioether

I


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subfamily P450LaMO, which was discovered in Labrenzia aggregata by genomic data mining, can convert prochiral sulfide derivatives also with high conversion and stereoselectivity.83 Significantly, recombinant E. coli (P450pyrI83H-GDH) coexpressing three-component P450pyrI83H monooxygenase and glucose dehydrogenase (GDH) was engineered for asymmetric sulfoxidations under an aqueous/ionic liquid biphasic system, giving higher (R)-enantioselectivity and higher specific activity than any other known P450-monooxygenases for this type of reaction (Scheme 22).84 Despite this progress, the application

varied, in which the biocatalysts prefer boron−carbon oxidation over Baeyer−Villiger oxidation or epoxidation (Scheme 24).91 Scheme 24. BVMO-Catalyzed Oxidation of BoronContaining Vinylic Compounds

Scheme 22. P450-Catalyzed Asymmetric Sulfoxidations under a Biphasic System

In addition, it was interesting to find that PAMO can also catalyze kinetic resolution of racemic organoborons (Scheme 25a) or organoseleniums (Scheme 25b) to produce chiralcentered boron compounds91 and chiral-centered selenium compounds,92 respectively.

of these P450 enzymes on a preparative scale and the extension of substrates spectrum (limit to aryl aliphatic sulfides) in the enantioselective sulfoxidations remain to be addressed. 2.3. Oxidation of Amines, Boranes, and Selenium Compounds

Scheme 25. PAMO-Catalyzed Kinetic Resolution of Racemic Organoboron and Organoselenium

85−88

In addition to sulfide, other heteroatoms such as nitrogen, boron,89−91 and selenium compounds89−93 can also be oxygenated, but they are less investigated compared to the enantioselective sulfoxidations. Some representative cases are displayed here, such as CHMOAcineto catalyzed asymmetric oxidations of secondary and tertiary amines to the corresponding amine N-oxides (Scheme 23a and b).86,87 Moreover, class B Scheme 23. Flavin-Dependent Monooxygenases-Catalyzed Oxidation of Amines

2.4. Hydroxylation of Arenes

2.4.1. Using Flavin-Dependent Monooxygenases as Catalyst. Direct hydroxylation of aromatic ring is very attractive in organic synthesis. Flavin-dependent monooxygenases are able to hydroxylate C−H bonds of electron-rich (hetero)arenes (aromatic molecules with substitutents such as a hydroxyl or an amino group) under mild conditions.94 According to the flavin-dependent monooxygenases-catalyzed mechanism, dioxygen interacts with reduced FADH2 and can form a reactive C4a-hydroperoxyflavin. This species serves as a weak electrophile, typically hydroxylating an aromatic substrate with activating substituents (−OH or −NH2 group) at ortho or para position. Within the active site of flavoproteins, the catalytic species C4a-hydroperoxyflavin can place the “OH+” equivalent regioselectively proximal to a specific site on the

flavin-dependent monooxygenases Aspergillus f umigatus siderophore (SidA), a N-hydroxylating monooxygenase (NMO), can catalyze the hydroxylation of the side-chain amino group and also stimulate a lot of mechanism studies (Scheme 23c).88 The applicability of BVMOs in the oxidation of organoboron has been investigated through testing the chemo- and enantioselectivities on various boron-containing aromatic and vinylic compounds. Several BVMOs were tested, such as PAMO, M446G PAMO mutant, HAPMO, and CHMOAcineto. Using different types of BVMO, the degree of chemoselectivity J


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azelaica HBP1 with bound 2-hydroxybiphenyl was determined.102 Further still, phenol hydroxylase (PheA1) has also exhibited some synthetic potential in the synthesis of catechols (entry 6, Table 2). 103 It should be noted that 2,6dihydroxypyridine (DHP) can be converted to the corresponding 2,3,6-trihydroxypyridine by DHP hydroxylase. This hydroxylation product spontaneously underwent oxidization and dimerization to form a blue pigment under the oxygen atmosphere (entry 7, Table 2).104 In addition, polyketide hydroxylases such as PgaE were reported to be involved in gaudimycin C biosynthesis in Streptomyces species (entry 8, Table 2).105 The p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii can catalyze the hydroxylation of various phenolic acids. The substitution of a residue close to the phenolic group binding site yields the S146A variant that is found to be more potent than the WT enzyme in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA) to produce 3-hydroxy-4-amino phenylacetic acid (3-OH-4-APA). It demonstrated that a single-site mutation at S146 can expand the substrate spectrum of HPAH to include an aniline compound (entry 9, Table 2).106 2.4.2. Using P450 Monooxygenases as Catalyst. P450monooxygenases are also capable of catalyzing hydroxylations of aromatic rings. The oxidation process is different from flavindependent monooxygenases, and the substrates scope is not limited to electron-rich (hetero)arenes.21 It is generally believed that the catalytic process involves the epoxidation of the aromatic ring, generating an arene oxide. This unstable species rapidly undergoes the epoxide ring opening and a hydride anion migration, affording a ketone intermediate, which subsequently tautomerizes to the final phenolic product (Scheme 27).107 Although the mechanism of the aromatic hydroxylation has been studied, there is no consensus mechanism that can reasonably explain all of the experimental results. Recently, Marcus plot analysis of aromatic hydroxylation reactions by compound I model complexes was conducted by Asaka and Fujii to study the mechanism. It was revealed that an electron transfer process is probably involved in the rate-limiting step. A new reaction mechanism was proposed in which the electron transfer between an aromatic substrate and FeIV(O)(porphyrin·+) is in equilibrium in a solvent cage and coupled with the subsequent C−O bond formation.108 The hydroxylation of polycyclic aromatic hydrocarbons (PAHs) is of great importance due to the environmental concerns of these compounds. Enzymatic biodegradation of these environmental contaminants is therefore highly attractive for synthetic chemists to turn the pollution into high-valued compounds. It should be noted that P450cam has been engineered for hydroxylation of PAHs.109 The structure and function of this enzyme has also been carefully studied.110,111 Another important aspect of enzymatic aromatic compounds hydroxylation is the drug metabolism.112,113 In addition, the P450s-catalyzed hydroxylation of simple arenes, such as o-, m-,114 and p-xylene,115 as well as tert-butylbenzene,116 were reported. Moreover, nonheme iron oxygenases were also found to be capable of hydroxylating aromatic rings.117,118 Recently, the selective benzene hydroxylation to synthesize phenol, without forming overoxidation products, is achieved by cytochrome P450BM3 with the assistance of amino acid derivatives. It was found that the use of N-heptyl-L-proline modified with L-phenylalanine (C7-L-Pro-L-Phe) 33 as decoy

electron-rich aromatic substrates. It subsequently undergoes electrophilic substitution of the aromatic C−H bond to produce the hydroxylated products (Scheme 26).95 Scheme 26. Electrophilic Attack by OH+ in FlavinDependent Hydroxylases

Flavoprotein monooxygenases (FPMOs) can be divided into six different subclasses. Single-component aromatic hydroxylases (class A) is the largest of the FPMOs subgroups, catalyzing almost exclusively hydroxylation of aromatic rings. They are the focus of this part. The mechanism of class A FPMOs includes substrate binding, the reductive and oxidative half reactions, and the utilization of FAD that is bound noncovalently and in high affinity. Some excellent reviews significantly cover this field, including the structures, function, and catalysis of the mentioned frequently used flavoenzymes.94−96 Herein, related examples of class A flavindependent monooxygenases catalyzed hydroxylation of electron-rich (hetero)arenes are reviewed and displayed in Table 2. Phenol hydroxylase was known to catalyze the hydroxylation of simple phenols to their corresponding o-diol derivatives. Several monosubstituted phenols are accepted as substrates, including the P-aminophenol (entry 1, Table 2).97 Moreover, the hydroxylation of 2,4-dihydroxybenzoate using PHBH from R. opacus 557 gave the 2,3,4-trihydroxybenzoate with good regioselectivity (entry 2, Table 2).98 The 3-hydroxybenzoate 4-hydroxylase (MHBH) from Comamonas testosteroni KH122-3s converts 3-hydroxybenzoate into 3,4-dihydroxybenzoate in the presence of NADPH and O2 (entry 3, Table 2).99 By using 3-hydroxybenzoate-6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 as the catalyst, regioselective hydroxylation of 3-hydroxybenzoate can produce the 2,5-dihydroxybenzoate (entry 4, Table 2).100 2-Hydroxybiphenyl 3-monooxygenase (HbpA) catalyzes the ortho-hydroxylation of various 2-substituted phenols using NADH and molecular oxygen. The HbpA has been utilized in the hydroxylation of 2-hydroxybiphenyl to 3-phenylcatechol in preparative scale (entry 5, Table 2).101 Recently, the structure of HbpA from the soil bacterium Pseudomonas K


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Table 2. Representative Examples of Flavin-Dependent Monooxygenases-Catalyzed Hydroxylation of Electron-Rich (Hetero)arenes

molecules can strongly activate P450 BM3 for benzene hydroxylation to generate phenol with the catalytic turnover rate and the total turnover number reaching 259 min−1 P450BM3−1 and 40 200 P450BM3−1, respectively. The coupling efficiency of this transformation reached up to 43% (Scheme 28).119 Further screening of carboxylic acids and amino acids as efficient decoy molecules is expected to enhance the catalytic activity of P450BM3. This work represents an attractive alternative for phenol production and shows that amino acid derivatives with a totally different structure from fatty acids can also be used as decoy molecules for aromatic hydroxylation by WT P450BM3.42 Selective hydroxylation of monosubstituted benzene is challenging in organic synthesis because a mixture of ortho-, meta-, and para-products could be produced. Remarkably, by using the engineered P450BM3 variant M2 (R47S/Y51W/

Scheme 27. Proposed Reaction Mechanism for Benzene Hydroxylation by P450s

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nice example that serves as a benign alternative for the production of human relevant metabolites.124

Scheme 28. P450BM3 Catalyzed Hydroxylation of Benzene to Phenol Using Amino Acid Derivatives As Decoy Molecule

Scheme 31. P450BM3 Variant-Catalyzed Selective Aromatic Hydroxylation of Meclofenamic Acid

I401M) as catalyst, the efficient and regioselective aromatic hydroxylation of six monosubstituted benzenes under mild conditions was reported by Schwaneberg and co-workers. The influence of substituents (CH3, OCH3, Cl, Br, I, and F) on regioselective hydroxylation was investigated. It was found that, for the substituents (CH3, OCH3, Cl, Br, and I) on the aromatic ring, the regioselectivities are excellent (generally >95%). Despite that, for the fluorobenzene, a 49:51 ratio of ortho-product/para-product was formed (Scheme 29).120 The power of this o-hydroxylation method is further demonstrated by the application in the direct one-pot synthesis of L-tyrosine derivatives.121

P450BM3 mutants can be used to regioselectively hydroxylate 17β-estradiol (E2) 37. By investigating the catalytic activity of a set of P450BM3 mutants with 17β-estradiol, the bacterial P450BM3 was found to be capable of catalyzing the same reaction as human P450s, generating 2-OH E2 38 as a major metabolite (Scheme 32).125 These results suggested that P450 BM3 mutants would be useful for the enzymatic hydroxylation of steroid hormones. Scheme 32. P450-Catalyzed Hydroxylation of 17β-Estradiol (E2)

Scheme 29. P450-Catalyzed Regioselective Aromatic Hydroxylation of Six Monosubstituted Benzenes

The daidzein 3′-hydroxylase P450 enzyme (3′-DH), which is responsible for the hydroxylation of daidzein 39 at the 3′position, was engineered into an artificial self-sufficient daidzein hydroxylase (3′-ASDH). This fusion enzyme is more potent in the hydroxylation of daidzein. Furthermore, a recombinant S. avermitilis host was developed for the expression of 3′-ASDH and the production of the hydroxylated product 40. The conversion reached 34.6% and was 5.2-fold higher than that using the WT enzyme (Scheme 33).126

The self-sufficient P450RhF (CYP116B2) from Rhodococcus sp. in a whole-cell system was used to catalyze the highly regioselective oxidation of diclofenac 34 to 5-hydroxydiclofenac 35. It demonstrates the potential for gram-scale production of human drug metabolites through recombinant whole-cell biocatalysis. 5-Hydroxydiclofenac and its subsequent oxidative metabolite are of particular interest for toxicology studies because they have been shown to provide an antigenic determinant for immune cell activation in mice (Scheme 30).122

Scheme 33. Selective Hydroxylation of Daidzein 39 by 3′ASDH

Scheme 30. P450RhF Catalyzed Regioselective Oxidation of Diclofenac to 5-Hydroxydiclofenac

Stilbenoids display versatile biological properties, and their demands have been increasing over the years. However, traditional methods for their access required tedious preparation. Recently, the cytochrome P450-monooxygenase 154E1 from Thermobif ida f usca YX (CYP154E1) was identified, and it enables the synthesis of (E)-4,4′-dihydroxystilbene 41 via direct double hydroxylation of (E)-stilbene 42. The construction of a triple mutant led to significant enhancement of catalytic efficiency versus the WT enzyme, and the substrates scope was further enlarged to include ortho- and meta-substituted hydroxystilbenes (Scheme 34).127 2.4.3. Using Tyrosinase As Catalyst. It should be mentioned that the well-studied binuclear copper enzyme tyrosinase (Ty), a monophenol monoxygenase, can catalyze the conversion of phenols to o-diphenols.128−130 Ty catalyzes the ortho-hydroxylation of substrate 43 to o-diphenol 44, and the

Meclofenamic acid 36 has been used for treatment of pain and inflammation. WT P450BM3 was tested for its ability to hydroxylate 36, but only very low substrate conversion (99% epoxidation selectivity at titers >250 mg L−1 by in vivo synthesis of 50 (Scheme 37).158 Scheme 37. Application of Engineered P450BM3 in the Semisynthetic Production of Artemisinin

Engineered P450BM3 can also catalyze enantioselective epoxidation of simple terminal alkenes. Two efficient P450BM3 variants were generated by Arnold and co-workers by using saturation mutagenesis and recombination coupled with a colorimetric high-throughput screening for the identification of efficient epoxidation catalysts. These variants convert various terminal alkenes to either (R)- or (S)-epoxide with high catalytic turnovers and epoxidation selectivities (Scheme 38).159 This work demonstrated that the P450BM3 active site could be readily molded for enantio- and regioselective oxidations. The development of efficient methods for the selective epoxidation of complex small molecules is highly attractive. These compounds have numerous different C−H bonds and functional groups, making them challenging targets for selective oxidation. Fasan and co-workers demonstrated that unnatural amino acid mutagenesis can serve as a promising strategy for improving the catalytic efficiency and regioselectivity of P450s, P


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Scheme 39. P450BM3 Mutant 139-3 Catalyzed Epoxidation of (+)-Nootkatone

active intermediate. Styrene and styrene derivatives react with this intermediate and are exclusively transformed into the corresponding (S)-epoxides. Meanwhile, an NADH-dependent reductase component (StyB) catalyzes the transfer of hydrogen between reduced nicotinamide cofactors and oxidized FAD.167 These two components have their own catalytically active sites, and the epoxidation abilities of StyA depend exclusively on FADH 2 . Because of the high cost and instability of nicotinamide cofactors NAD(P)H, an efficient electrochemical regeneration of FADH2 strategy has been developed.168 StyA required FADH2 for catalysis, but the electron transport chains are rather complicated, which to some extent complicates the practical application of these enzymes.9 Recently, a more simplified and efficient FADH2 regeneration system was developed by utilizing the nicotinamide cofactor mimic 1benzyl-1,4-dihydronicotinamide (BNAH) as the sole reductant. By using the cheap and efficient BNAH as a stoichiometric reducing agent, preparative-scale biocatalytic asymmetric epoxidation and sulfoxidation can be efficiently performed to produce the corresponding enantiopure epoxides and sulfoxides.169 Despite isolated SMO being successfully applied in combination with enzymatic NADH regeneration,170 the process based on E. coli whole cells expressing those SMOs has been proven superior in consideration of the limited stability of cell-free enzymes, and it has therefore been applied in the majority of reported studies. Most SMOs are capable of converting native styrene substrate to (S)-styrene epoxide with excellent enantioselectivity.144 However, substrates with electron-withdrawing and bulky groups were not accepted in early work.171 Significantly, by using the SMOs from bacteria Rhodococcus sp. ST-5 and ST-10 expressed in E. coli, the corresponding (S)-epoxides can be produced with high stereoselectivity from styrene derivatives, halogenated styrene derivatives, and even short-chain 1-alkenes (Scheme 45).172 Moreover, by employing an efficient catalytic system containing recombinant E. coli cells expressing Rhodococcus sp. ST-10 (RhSMO) and Leifsonia sp. alcohol dehydrogenase (LSADH) in the aqueous/organic biphasic reaction conditions, various alkenes, including styrene derivatives, and vinylpyridines can also be stereoselectively epoxidated to afford

Scheme 40. WT P450BM3 Catalyzed Epoxidation of Substrate 54

Scheme 41. Predictable Stereo- and Chemoselective Epoxidations with P4503A4

Moreover, by using P450BM3 mutants V78A/F87A, the preparative scale epoxidation of β-cembrenediol 59 at C7C8 double bond can be achieved in >99:1 diastereomeric ratio (dr), albeit in low isolated yield (Scheme 44).166 2.5.1.2. Asymmetric Epoxidation of Vinyl Aromatic Compounds. 2.5.1.2.1. Using Flavin-Dependent Monooxygenases. Enantiopure styrene oxides are important building blocks in medicinal chemistry. The frequently used enzymes for the epoxidation of styrenes and its derivatives are flavin- and heme-dependent monooxygenases. Flavin-dependent monooxygenases are capable of epoxidizing activated, vinyl aromatic CC double bonds.27 Styrene monooxygenase (StyAB) from Pseudomonas sp. strain VLB120 was one of the most wellstudied enzymes of this class. This is a two-component enzyme containing a FADH2-dependent oxygenase component (StyA); it binds tightly to the reduced FAD to form the StyA−FADred complex, which activates O2 to yield the FAD C4a-peroxide

Scheme 42. Altering the Selectivity of Cytochrome P4503A4 by Active-Site Crowding

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Scheme 43. MycG-Catalyzed Selective Epoxidation of Substrate 57

Scheme 44. P450BM3 Catalyzed Epoxidation of β-Cembrenediol 59

Scheme 45. SMOs-Catalyzed Epoxidation of Styrene Derivatives, Halogenated Styrene Derivatives, and ShortChain 1-Alkenes

Scheme 46. SMOs-Catalyzed Asymmetric Epoxidation of Allylic Alcohols

Scheme 47. SMOs-Catalyzed Kinetic Resolution of Substrate 63

(S)-epoxides with good yields. The LSADH is in charge of regenerating NADH utilizing 2-propanol and supplies NADH to the SMO for the epoxidation reaction.173 In addition to styrene, SMOs can also catalyze the asymmetric epoxidation of primary allylic alcohols. For example, alcohol 60 can be epoxidized by recombinant E. coli employing the SMO from the Pseudomonas species and afforded the corresponding (2S,3S)-epoxide 61 with >99% ee (Scheme 46a).174 Furthermore, epoxidation of conjugated secondary allylic alcohols 62 also proceeded smoothly with SMO and could be achieved with excellent stereoselectivity (Scheme 46b).174 Remarkably, SMO also found applications in the kinetic resolution of secondary phenyl allylic alcohols. The kinetic resolution of racemic 1-phenylprop-2-enol 63 can be performed by utilizing the whole cells of recombinant E. coli expressing the SMO from Pseudomonas sp. LQ26, producing 64 with >99% ee and 98% de along with the recovery of the (R)-alcohol 65 with >99% ee (Scheme 47).175 Recently, an enzymatic one-pot, twostep cascade reaction was performed by Wu and co-workers by using prochiral α,β-unsaturated ketones as the substrates. This reaction showed significant improvement over the use of SMO alone. It contains asymmetric bioreduction of α,β-unsaturated

ketones and subsequent SMO-catalyzed epoxidation of CC double bond.176 Very recently, Liu and Wu reported a direct asymmetric epoxidation of α,β-unsaturated ketones 66 for access of α,βepoxyketones 67 by using recombinant E. coli coexpressing alcohol dehydrogenase from Rhodococcus erythropolis DSM 43297 (READH) and SMO as catalyst. This reaction proceeded efficiently, and the corresponding products are generally obtained in high yields as well as high stereoselectivities. It overcomes the difficulty of direct bioepoxidation of electron-deficient olefins, which is more challenging than the general or electron-rich olefins. The products α,β-epoxyketones 67 can subsequently undergo enzymatic reduction to afford the optically pure allylic epoxy alcohols (Table 3).177 R


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for the efficient preparation of enantioenriched (S)-epoxyalkanes is viable. The biocatalytic system employed molecular oxygen as oxidant and 2-propanol as a hydrogen donor, and various aliphatic alkenes (less than C9) were epoxidized with good to excellent enantioselectivities.179 Moreover, by utilizing this RhSMO expressed in the organic solvent-tolerant microorganism Kocuria rhizophila DC2201, optically pure straightchain (S)-epoxyalkanes can be produced from linear terminal alkenes in an organic solvent−water biphasic reaction system using glucose and molecular oxygen.180 2.5.1.2.2. Using Other Monooxygenases. Cytochrome P450-monooxygenase can also catalyze the epoxidation of styrenes to prepare chiral styrene oxide derivatives. However, the stereoselectivities of native P450s are generally inferior.147 Protein engineering could serve as a promising tool to improve P450-monooxygenase catalysis. Some engineered P450s display good stereoselectivity in biocatalytic epoxidation reactions. For examples, residue size at position 87 of P450BM3 was found to have a strong impact on determining the stereoselectivity of epoxidation. The F87G mutant exhibited much higher selectivity than the wild-type, or F87 V and F87A mutants, leading to (R)-styrene oxide and (R)-3-chlorostyrene oxide with 92% and 94.6% ee, respectively.181,182 P450cam from P. putida has been utilized as a styrene epoxidation catalyst, particularly when using the Y96F mutant; significantly improved activity and selectivity for styrene epoxidation was observed compared to the WT enzyme.183 Cytochrome P4503A4 is the major human P450 responsible for the metabolism of carbamazepine (CBZ). Engineered P4503A4 mutants I369F, I369L, A370V, and A370L can lead to increased turnover of CBZ to the corresponding 10,11-epoxide product.184 Recently, by using the resting cells of E. coli expressing a triple mutant of P450pyrTM, epoxidation of several parasubstituted styrenes 71 was achieved with excellent (R)selectivity (up to 99.5% ee) and high conversion (82−97%). Interestingly, using this enzyme, the epoxidation of orthosubstituted styrenes 72 and 1,1-disubstituted alkenes 73 are also effective, providing the (S)-selective products with high ee (Scheme 49).185 P450tol monooxygenase from Rhodococcus coprophilus TC-2, which was coexpressed with the ferredoxin and ferredoxin

Table 3. READH- and SMO-Catalyzed Direct Asymmetric Epoxidation of α,β-Unsaturated Ketones

entry

R

HPLC yield (%)

ee (%)

1 2 3 4 5 6 7 8 9 10

H o-F m-F p-F o-Cl m-Cl p-Cl o-Br m-Br m-Me

>99 >99 >99 >99 92 >99 95 67 93 >99

>99 >99 >99 >99 >99 >99 96 >99 >99 >99

One-pot cascade biocatalysis is important in organic synthesis. The intracellular epoxidation and hydrolysis cascade of aryl olefins 68 represents an efficient enantioselective dihydroxylation method for the preparation of chiral vicinal diols. E. coli (SSP1) coexpressing SMO combined with epoxide hydrolase SpEH were used as catalyst for (S)-enantioselective dihydroxylation, giving (S)-vicinal diols 69 in high stereoselectively. Combining SMO with epoxide hydrolase StEH displaying different regioselectivities furnished (R)-enantioselective dihydroxylation product 70. This approach developed by Li and co-workers is a rare example that reversed the overall enantioselectivity of cascade reaction by simply changing the regioselectivity of a single step. This method is complementary to well-developed Sharpless dihydroxylation, accepting cisalkenes as substrates and performing enantioselective transdihydroxylation (Scheme 48).178 It should be mentioned that, in addition to styrene and styrenecderivatives, some SMOs can also be used to epoxide aliphatic alkenes. By using RhSMO together with LSADH, the enantioselective epoxidation of straight-chain aliphatic alkenes Scheme 48. Preparation of Chiral Vicinal Diols via the Intracellular Epoxidation and Hydrolysis Cascade

Scheme 49. Engineered P450pyr Monooxygenase-Catalyzed Asymmetric Epoxidation of Alkenes

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reductase from Sphingomonas sp. HXN-200, and a GDH in E. coli T7 displaying unique and excellent enantioselectivity toward several ortho- and meta-substituted styrenes furnished the (R)-epoxidation products in up to 99.7% ee and (S)epoxidation of two para-substituted styrenes with 90.2% and 90.5% ee, respectively (Scheme 50).186 The high efficiency of P450tol monooxygenase makes it an ideal catalyst for the preparation of these useful and valuable pharmaceutical intermediates.

The oxidative cleavage of CC bonds contains the cleaving of aliphatic and aromatic CC double bonds. The most frequently used enzymes are iron-dependent dioxygenases. Indole amine 2,3-dioxygenase (IDO) and tryptophan 2,3dioxygenase (TDO) are heme-dependent dioxygenases that catalyze cleavage of CC double bonds of the pyrrole ring of tryptophan 74 to afford N-formylkynurenine 75 (Scheme 52); their catalytic properties and mechanism have been welleluciated.190,191

Scheme 50. P450tol Monooxygenase-Catalyzed Asymmetric Epoxidation of Substituted Styrenes

Scheme 52. TDO- or IDO-Catalyzed Cleavage of CC Double Bonds of Tryptophan

Nonheme iron-dependent enzymes require O2 for alkene cleavage. One important feature is that they can often differentiate between various CC double bonds within one molecule, even from many conjugated double bonds, which is not accessible by use of chemical methods. Because the detailed description of nonheme Fe/O2 catalysis is covered by another article in the “Oxygen Reduction and Activation in Catalysis” thematic issue of Chemical Reviews, only some representative cases are presented here. For example, carotenoid cleavage dioxygenases (CCDs) can selectively cleave CC double bonds of carotene 76 to produce apocarotenoids 77 (Scheme 53a).192 The AtCCD1 from Arabidopsis thaliana cleaves various carotenoids 78 symmetrically at the 9,10 and/or 9′,10′ double bond (Scheme 53b).193 Isoenzymes of the lignostilbene-α,βdioxygenase (LSD) from Sphingomonas paucimobilis transform astilbene-type substrate 79 selectively (Scheme 53c).194 Stilbene oxygenases from Novosphingobium aromatic ivorans DSM12444 (NOV1 and NOV2) selectively cleave transstilbene-type substrates 80 (Scheme 53d).195 Fe(II)-dependent β-diketone dioxygenase from Acinetobacter johnsonii (Dke1) is also reported to cleave the enol form of the 2,4-pentanedione 81 (Scheme 53e).196 Enzymes can also cleave aromatic CC bonds. The selective cleavage of catechols and substituted catechols CC double bonds can be performed by the catechol dioxygenases. These dioxygenases require a mononuclear iron center without cofactors.138,197 There are two types of cleavages that have been found for catechol: one is the cleavage of the bond between the two hydroxyl groups to yield muconic acid derivatives; the other is the cleavage of one bond neighbored and one carbon with a hydroxyl group to form 2hydroxymuconaldehyde. Moreover, some Cu-, Mn-, and Nidependent enzymes have also been found to be effective in the cleavage of catechol CC double bonds.189

Moreover, the well-studied cyclohexanone monooxygenase CHMOAcineto from Acinetobacter calcoaceticus NCIMB9871 is able to catalyze the epoxidation of Michael acceptor-type alkenes. Numerous olefins were tested, but only dimethyl- and diethyl vinyl phosphonate were accepted as substrate by CHMO. Despite the narrow substrate scope, this represents the first example of enantioselective epoxidation mediated by cyclohexanone monooxygenase (Scheme 51).187 Scheme 51. CHMOAcineto Catalyzed Epoxidation of Vinyl Phosphonate

2.5.2. Oxidative Cleavage of CC Double Bonds. One of the most investigated reactions in organic synthesis is the cleavage of CC double bonds to form the corresponding carbonyl products, especially in the incorporation of oxygen functionalities into molecules, and the removal of protecting groups as well as tailoring of large molecules.188 Compared to the chemical methods, the enzymatic strategies enjoy the advantages of milder reaction conditions and display good chemo- and regioselectivity.189 Some representative examples of heme and nonheme iron-dependent enzymes catalyze cleavage of CC double bonds using molecular oxygen as oxidant are grouped here.

2.6. Oxygenation of sp3 C−H Bonds

Selective enzymatic hydroxylation of nonactivated C−H bonds is of particular importance because of the prevalence of hydrocarbon compounds and relative scarcity of their chemical alternatives. Among the various enzymes employed, P450monooxygenases have attracted significant attention due to their potent abilities in the direct hydroxylation of nonactivated C−H bonds. All of the bacterial CYPs characterized so far that T


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protein and its properties to meet our needs have significantly expanded the utilities of P450s for hydrocarbon oxygenations, with significant contributions made by the groups of Reetz and Arnold.199−202 The utilization of P450-monooxygenases in the selective hydroxylation of nonactivated C−H bonds has been significantly reviewed.9,21,203−207 It should be mentioned that the long-chain degrading monooxygenase LadA can also catalyze the oxidation of alkanes from C15 to C36 to their corresponding terminal alcohols. So far, this is the very rare flavin-dependent monooxygenase reported that is capable of hydroxylating nonactivated hydrocarbons.208,209 Proline hydroxylases, which belong to the family of iron(II)- and 2oxoglutarate-dependent nonheme dioxygenases that hydroxylate L-proline and derivatives, have increased in popularity in recent years.210,211 This part provides an updated overview of this exciting and rapidly growing area of chemistry with a particular focus on the current state of the art of engineering or state of use for preparative synthesis using P450-monooxygenases. 2.6.1. Selective Hydroxylation of Structurally Simple Molecules. Methane, with the highest bond strength of any alkane (104.9 kcal mol−1), remains a big challenge in the direct C−H hydroxylation.212,213 Tremendous progress has been made in the past few decades. Particularly, a recent comprehensive review, “Alkane Oxidation: Methane Monooxygenases, Related Enzymes, and Their Biomimetics”, significantly covers this field. Very detailed description of the frequently used enzymes, including the soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO), have been presented. 214 Moreover, insightful perspective of this significant area has also been presented. The structure, the reaction mechanism, and the biological methane-oxidizing systems have been well-summarized.215 Thus, little update on this field is needed here. Cytochrome P450-monooxygenases are capable of oxidizing less-reactive C−H bonds; however, gaseous alkanes are not accepted for WT P450s.216 This is because of the relatively large binding pockets of CYPs; small compounds do not have a statistically high enough probability to be properly oriented near the oxyferryl moiety for rapid oxidation. Elegant work was reported in 2011 by Reetz and co-workers, who first introduced the addition of appropriate chemically inert perfluorinated carboxylic acids (PFCs) to the enzyme, allowing small alkanes such as propane (but not methane) to be oxidized with

Scheme 53. Representative Examples of Non-heme IronDependent Enzymes-Catalyzed Cleavage of CC Double Bonds

are capable of oxidizing alkanes are members of the CYP153 family of soluble cytochrome P450s.198 The reactions catalyzed by naturally occurring P450s are not always satisfactory. Protein engineering, particularly by directed evolution, has emerged as a powerful tool in creating robust enzymes with improved catalytic performance. The abilities to modify or engineer a

Figure 7. Crystal structure of PFC9-L-Trp-bound P450BM3 (left, PDB code 3WSP); a plausible catalytic cycle of the P450BM3 decoy-molecule system (right). U


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Figure 8. Mutagenesis of amino acids located in the binding pocket (yellow area) and substrate access channel (blue area) of the protein. Reprinted with permission from ref 222. Copyright 2016 Wiley-VCH.

pronounced enzyme activity.217 This concept has also been independently reported by the Watanabe group in the oxidation of propane and butane.218 Despite this elegance, however, the interaction of PFCs with P450BM3 may not be sufficient enough to fully exploit the catalytic potential of P450BM3. As reported by Shoji, Watanabe, and co-workers, the use of N-perfluoroacyl amino acids as decoy molecules strongly activates the WT P450BM3, and drastically enhanced activities in the hydroxylation of propane were observed.219 The hydroxylation rate of propane reached to 256 min−1 P450−1 by employing N-perfluorononanoyl-L-leucine as a decoy molecule and was 4 times higher than that of PFC10 (67 min−1 P450−1).218 Furthermore, the crystal structure of N-perfluorononanoyl-L-tryptophan (PFC9-L-Trp)-bound P450BM3 was successfully obtained. A DMSO molecule was observed at the sixth ligand position of the heme instead of a water molecule. In consideration of the similar size of DMSO with small alkanes, the structure to some extent can be regarded as a small alkanebound P450BM3 model. From the crystal structure, it was also observed that the terminal perfluoromethyl group of PFC9-LTrp is situated close to the DMSO molecules, indicating that the role of decoy molecules may be to partially occupy the substrate-binding site of P450BM3 and reform the active-site pocket, so that the small molecules can be accommodated for catalysis (Figure 7).219 Compared to time-consuming protein engineering, these strategies simply require the addition of an appropriate chemically inert perfluoro fatty acid or amino acid to the enzyme to trigger a strong activating effect, constituting a powerful tool in expanding the utility of P450BM3 for C−H bond oxygenations.220 The development of highly efficient methods for hydroxylation of terminal C−H of fatty acids is important.221 CYP153AM.aq.-CPRBM3 can selectively hydroxylate at terminal positions of medium- and long-chain fatty acids, but the activity needs further improvement. To address the low activity of CYP153AM.aq.-CPRBM3 toward these fatty acids, diverse mutant libraries were generated. Successive mutagenesis involving positions in the binding pocket (yellow) and the substrate access channel (blue) demonstrate successful synergistic interactions (Figure 8). The combination of the most active single variants leads to the identification of an improved enzyme. By using a double variant G307A/S233G, the activity for the terminal hydroxylation of medium-chain-length fatty acids increased 2-fold relative to the WT enzyme.222

Moreover, cytochrome P450BM3 catalyzed regioselective hydroxylation of C12−C15 terminal fluorinated fatty acids was reported by Yu and co-workers. The products from the terminal methyl fluorinated fatty acids were largely hydroxylated at the ω-3 positions (Scheme 54).223 The effects of Scheme 54. Regioselective Hydroxylation of Terminal Methyl Fluorinated Fatty Acid by WT P450BM3

fluorinated terminal methyl groups on the conversion of C12− C15 fatty acids by cytochrome P450BM3 were examined. A lower Michaelis−Menten constant (Km) indicated that fluorinated fatty acids are more suitable substrates than the native fatty acids. The terminal fluorinated methyl group introduces additional interactions of the substrate with the amino acid residues in the hydrophobic pocket, leading to improved regioselectivity. Recently, P450pyr, a terminal-selective cytochrome, has been successfully engineered for the enantioselective and subterminal-selective hydroxylation of alkanes. A sensitive colorimetric high-throughput screening (HTS) assay was developed by Li and co-workers to measure both the regio- and enantioselectivity of the hydroxylation reaction. By using this HTS assay and ISM technique, P450pyrSM1(A77Q/I83F/N100S/F403I/ T186I/L302V) was created for the hydroxylation of n-octane 82, which produces (S)-2-octanol 83 with 98% ee and >99% subterminal selectivity. This is the first enzyme reported for this type of highly selective alkane hydroxylation. Another sextuple mutant P450pyrSM2 (I83F/N100S/T186I/L251V/L302V/ F403I) catalyzed the hydroxylation of propylbenzene 84 to afford (S)-1-phenyl-2-propanol 85 with 95% ee and 98% subterminal selectivity (Scheme 55).224 The evolution of P450pyr hydroxylase turns its terminal selectivity fully into subterminal selectivity, which could provide more inspiration in guiding the engineering of other P450 enzymes for selective oxygenations. Significantly, integrating CYP154A8 along with suitable redox partners into a whole-cell system is effective for the V


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Scheme 55. Engineering of P450pyr Hydroxylase for Hydroxylation of Alkanes

Scheme 56. Possible Products of P450BM3 Catalyzed Hydroxylation of Methylcyclohexane

genesis experiments were carried out. Upon screening a total of 760 transformants, it was found that variant F87A produced the undesired tertiary alcohol 88 in 71% regioselectivity due to the hydroxylation at the weakest C−H bond, while variant A328F afforded the cis-(1S,2R)-87 in 71% regioselectivity, 97% diastereoselectivity, and enhanced enantioselectivity (92% ee). The presented prochiral monosubstituted cyclohexane derivatives desymmetrization catalyzed by WT P450BM3 or mutants thereof represents the first strategy for accessing highly enantiopure vicinal disubstituted cis-cyclohexanols via a single C−H activation step.226 Chiral allylic alcohols of ω-alkenoic esters are useful building blocks, but traditional methods are difficult to synthesize via direct stereoselective C−H hydroxylation. By using P450BM3 double mutant A74G/L188Q as a robust and selective biocatalyst, the asymmetric allylic hydroxylation of readily available ω-alkenoic esters 89 was reported by Pietruszka and co-workers. This method provided (S)-configured allylic alcohols 90 with high chemo- and enantioselectivity. Mechanistic studies revealed that the carbonyl moiety is crucial for the chemo- and enantioselectivity (Scheme 57). This P450BM3 mutant-catalyzed allylic C−H hydroxylation of linear terminal olefins gives the highest enantioselectivity observed to date.227

production of chiral 2-alkanols starting from alkanes. Both recombinant E. coli and P. putida whole-cell biocatalysts are able to produce chiral alkanols. The optimized P. putida whole-cell system yielded (S)-2-octanol with 87% ee from octane. Additionally, a combined P450−alcohol dehydrogenase (ADH) system was established to improve the enantiopurity of the desired 2-alcohols. By adding an ADH into this system, 5.4 mM (S)-2-octanol with 97% ee was obtained, and the achieved concentration of chiral 2-octanol is the highest reported for a P450-based whole-cell system so far (Figure 9).225

Scheme 57. P450BM3 Catalyzed Asymmetric Hydroxylation of ω-Alkenoic Esters Figure 9. CYP154A8-catalyzed regio- and stereoselective hydroxylation of alkanes to synthesize (S)-2-alcohols in a whole-cell system. Reprinted with permission from ref 225. Copyright 2016 Wiley-VCH.

To create two or more new chiral centers simultaneously upon a single oxidative hydroxylation of achiral substrates is challenging yet highly attractive. Exploring catalysts that enable the control of regio-, diastereo-, and enantioselectivity is a challenge. Reetz and co-workers demonstrated that such control is possible by using wild-type or mutant forms of the P450BM3 as catalysts in the oxidative hydroxylation of methylcyclohexane 86 and several other monosubstituted cyclohexane derivatives. Using the methylcyclohexane 86 as the model compound, hydroxylation at the two methylene units flanking the CH3-bearing carbon atom provides four possible products, cis-(1S,2R)-87, cis-(1R,2S)-87, trans-(1R,2R)-87, and trans-(1S,2S)-87, with regioisomers resulting from hydroxylation of other carbon atom also being possible (Scheme 56). Surprisingly, WT P450BM3 catalyzed the hydroxylation of methylcyclohexane with 78% regioselectivity, over 97% diastereoselectivity and 82% enantioselectivity in favor of cis(1S,2R)-87. To improve the enantioselectivity in the regioselective formation of cis-(1S,2R)-87, saturation muta-

The control of site-selectivity among similar C−H bonds is one of the key issues in developing C−H oxidation catalysts. Take the regioselective C7-hydroxylation of limonene as an example. In this compound, there are many reactive positions (two terminal allylic positions and three allylic ring positions for hydroxylation, and two double bonds for epoxidation). The WT enzyme converted (4R)-limonene 91 to four different oxidation products, but no terminal hydroxylation at the allylic C7 was observed. Pleiss and co-workers elegantly reported a generic strategy to engineer the P450BM3 into a selective catalyst capable of hydroxylating exclusively at the chemically less reactive, primary C7 position of (4R)-limonene 91 in up to 97% selectivity (Scheme 58).228 Directed evolution of the P450BM3 monooxygenase utilizing ISM at and near the binding site can be applied to generate P450 mutants that enable a high degree of both regio- and W


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Scheme 58. P450 BM3 Mutants Catalyzed C7-Hydroxylation of (4R)-Limonene 91

Scheme 61. P450BM3 Mutant M02 Enzyme-Catalyzed Hydroxylation Reaction

enantioselectivity in the oxidative hydroxylation of a given molecule via a single C−H oxidation step. Reetz and coworkers have shown that, by directed evolution, both (R)- and (S)-mutants of P450BM3 were evolved in the hydroxylation of cyclohexene-1-carboxylic acid methyl ester 92 to obtain (R)alcohol 93 and (S)-alcohol 94, respectively (Scheme 59). Meanwhile, WT P450BM3 only showed 84% regioselectivity (16% other oxidation products) and poor enantioselectivity in slight favor of (R)-alcohol 93 (34% ee).229

allylic sites as potential hydroxylation positions. The F87A/ I263L mutant enabled the regioselective hydroxylation of the neighboring positions C9 of β-cembrenediol 100 with 100% regioselectivity and an 89:11 dr. Meanwhile, the L75A/V78A/ F87G mutant led to 97% regioselectivity and a 74:26 dr of C10 hydroxylation product 101 (Scheme 62).166 Furthermore, a Scheme 62. P450BM3 Variants Evolved for the Selective Hydroxylation of β-Cembrenediol

Scheme 59. P450 BM3 Mutants Catalyzed Enantioselective Hydroxylation of Substrate 92

2.6.2. Selective Hydroxylation of Complex Molecules. Selective hydroxylation of complex organic molecules is challenging because different kinds of C−H bonds often coexist with each other as well as with other functional groups. Reetz and co-workers have shown that the control of regio- and stereoselective C−H bond hydroxylation of complex organic compounds such as steroids can be met by directed evolution of P450 enzymes. Testosterone 95, which is not accepted by WT P450BM3, was chosen as the model substrate in the oxidative hydroxylation reaction. However, using P450BM3 mutant F87A in the hydroxylation of testosterone, about 1:1 mixture of the respective 2β-96 and 15β-97 alcohols were delivered. By applying iterative rounds of the saturation mutagenesis technique, the mutants obtained could produce either of these two regioisomers with excellent regio- and diastereoselectivity (Scheme 60). Moreover, some of the best mutants can be applied to hydroxylate other steroidal substrates without additional mutagenesis. This work provides a useful toolbox that synthetic chemists can utilize for regio- and stereoselective hydroxylation of steroids.230 Furthermore, cytochrome P450BM3 mutant M02 can be applied for whole-cell biotransformation of a 17-ketosteroid norandrostenedione 98. Purified P450BM3 mutant M02 hydroxylated this steroid with >95% regioselectivity to form 16-β-OH norandrostenedione 99 (Scheme 61).231 Several P450BM3 variants were evolved for the selective hydroxylation of β-cembrenediol. This compound carried seven

P450BM3 mutant-catalyzed, one-pot, two-step hydroxylation of the β-cembrenediol 100 was recently developed. Successive hydroxylations catalyzed by the regioselective P450BM3 mutants F87A/I263L and V78A/F87G yielded the epimeric (9S,10R/ S)-β-cembrenetetraols with a 48:52 dr.232 The CYP101B1 monooxygenase from Novosphingobium aromaticivorans DSM12444 binds norisoprenoids with higher affinity than monoterpenoids and oxidized these substrates with excellent regioselectivity. The sesquiterpene lactone (+)-sclareolide 102 was stereoselectively hydroxylated by CYP101B1 to (S)-(+)-3-hydroxysclareolide 103 as the single product. The ester functional group of the substrate mimicked the carbonyl moiety of norisoprenoids and can be used as a directing group to anchor the monoterpenoid acetates in the active site of CYP101B1 tightly for oxidation, thus resulting in significant improvements in both activity and selectivity (Scheme 63).233 This is an elegant example demonstrating the use of a substrate engineering strategy. It showed that the simple addition of an ester group could be utilized as a chemical auxiliary or directing group to facilitate and control substrate binding, leading to

Scheme 60. P450-Catalyzed Regio- and Stereoselective Hydroxylation of Steroids

X


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and a carboxylate group within the active site (Figure 10).234 The amine acts as an anchor, essentially holding the substrate in a specific location, while the sugar serves as a linker, controlling the specific orientation of the substrate with respect to the active site. Thus, the site selectivity results from the combined effects of the active site, anchor, and linker. By harnessing this unique anchoring functionality, a variety of desosamine-containing unnatural substrates were synthesized and tested for oxidation with PikC. An engineered P450 enzyme PikCD50N-RhFRED was generated and was found to be ∼13-fold more active than the WT PikC. Using the PikCD50NRhFRED as a catalyst, macrocyclic substrates were hydroxylated to produce multiple products with moderate yields; however, no reaction occurs with substrates of smaller cores (Figure 11).236 This is probably due to the ring of the substrate

Scheme 63. CYP101B1 Monooxygenase-Catalyzed Selective Hydroxylation of the Sesquiterpene Lactone (+)-Sclareolide

selective and rapid oxidation of nonactivated C−H bonds in terpenoid frameworks. Another important case to be highlighted here is the utility of the cytochrome P450-monooxygenase PikC, from the pikromycin biosynthetic pathway. PikC hydroxylated its natural substrates 12-membered-ring macrolide 104 (YC-17) and 14membered-ring macrolide narbomycin 105 (Scheme 64).234,235 Scheme 64. PikC-Catalyzed Hydroxylation of YC-17 and Narbomycin

Figure 11. PikCD50N-RhFRED-catalyzed hydroxylation of desosaminecontaining unnatural substrates.

being too small to span the distance between the anchoring carboxylate group and the active iron-oxo species of PikC for catalysis. Although desosamine proved to be an effective anchoring group for unnatural substrates, the requirements of multiple synthetic steps for its preparation and the harsh conditions for its removal limit its utility; the development of a simplified anchoring group capable of interacting with the carbonate residue within the active site of PikC to promote C−H hydroxylations is thus highly attractive. Sherman, Montgomery, Podust, and co-workers showed that the desosamine in

It displayed unusually high substrate promiscuity, which is rarely found in secondary metabolic pathways. This level of substrate promiscuity could be explained by the mechanism in which natural substrates bind within the PikC active site. The cocrystal structure of PikC with YC-17 clearly revealed saltbridge interactions between the dimethylamino group of YC-17

Figure 10. Cocrystal structure of PikC with YC-17 (PDB code 2CD8) depicting the salt-bridge interactions between the dimethylamino group of YC-17 with E94. Y


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Scheme 65. PikCD50N-RhFRED-Catalyzed Oxidation of YC-17 Analogues (Ratios of C10:C12 Hydroxylation Are Given, and the Major Products Are Shown)

substrate YC-17 could be replaced with more accessible anchoring moieties. After a series of assessments of artificial linkers with lengths and geometries different from those of the native linker, it was observed that, by varying the anchoring auxiliary from 3-(dimethylamino)propanoic acid to meta(dimethylamino)benzoic acid, the selectivity of oxidation can be tuned to favor either C12 (3-fold) or C10 (20-fold) (Scheme 65).237 It was proposed that the more rigid anchoring group restricts the conformational freedom of the substrate within the active site, thus leading to the enhancement of regioselectivity. This work demonstrates that the utility of substrate engineering can serve as an orthogonal approach to protein engineering for efficient C−H hydroxylation of complex organic molecules. Enabled by advances in computing power, MD simulations can now serve as a powerful tool in providing insightful guidance in protein and substrate engineering. The efficiency and selectivity of PikC-catalyzed hydroxylation of a given substrate was iteratively evaluated through QM and MD calculations, with excellent correlation to experimental data. The information gained from these studies has already begun to make a significant impact on research. As illustrated in Figure 12, the substrate scope of PikC monooxygenase has been expanded from its natural 12- and 14-membered macrolides to a range of non-native substrates of small-ring scaffold.238 This tool will prove invaluable in the discovery of potent enzymatic systems.

Figure 12. Regioselective C−H hydroxylation of various cycloalkanes with PikC-RhFRED.

BVMOs is limited. Otherwise these are enzymes that are currently employed in preparative synthetic strategies. Researchers have tried pressurized reactors (air or O2) as well as bubbling O2 into reactions, which leads to foaming. Both can result in enzyme denaturing/inactivation. (2) The substrate scope is narrow. For example, the styrene monooxygenases have a very narrow substrate scope (only styrene analogues) and are therefore less frequently employed in synthetic routes. (3) Cytochrome P450 reactions are plagued by rapid catalyst decompositions, poor solvent tolerance, requirement for low substrate concentrations, and inherent enzyme instability at room temperature in solution. Besides, substrate concentrations must remain significantly lower than typical enzymatic reactions, which leads to substrate-dependent irreversible catalyst inactivation. A core problem with P450s is the generation of reactive oxygen species (ROSs) because of the decoupling of reducing equivalents into the cycle (NADPH) versus product hydroxylation. These ROSs lead to rapid catalyst decomposition.239 The poor solvent tolerance and instabilities can be addressed by protein engineering to deliver robust engineered variants. In addition to protein engineering, substrate engineering can also be applied in developing general methods for regio- and

3. SUMMARY AND OUTLOOK Enzymatic catalysis has been used as an environmentally friendly strategy to oxygenate organic compounds. This is especially because of the ever-increasing rate of the discovery of novel enzymes and the generation of more robust biocatalysts that significantly broaden the synthetic toolbox of organic chemists. The high activity and selectivity of enzymes is promising for streamlined molecule synthesis and late-stage modification of complex organic molecules. However, to make biocatalytic oxygenation truly efficient and practical, there are still a range of issues that need to be addressed. Below are some limitations that hinder these enzymes from being employed in preparative organic synthesis: (1) Mass transfer of oxygen is generally limited in these reactions. For example, the mass transfer of oxygen of the widely employed Z


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ACKNOWLEDGMENTS Financial support from the National Natural Science Foundation of China (nos. 21632001 and 21772002), National Basic Research Program of China (973 Program) (Grant no. 2015CB856600), National Young Top-Notch Talent Support Program, and Peking University Health Science Center (no. BMU20160541) is greatly appreciated.

stereoselective oxidation; some elegant works have been described in this contribution. Moreover, enzyme activities can also be enhanced by a combination of enzyme with dummy molecules such as perfluorinated carboxylic acids or amino acids to reform the enzyme active site. The hydroxylation of small alkanes such as propane and ethane has proven to be effective, but methane hydroxylation is still difficult to achieve with notable efficiency. The optimization of the structure of the decoy molecules is expected to further improve the hydroxylation activities of the WT P450BM3, making this continue to be an exceedingly interesting area of investigation. On the whole, the synthetic potential of oxygenases catalysis is immense, and it still has a lot to offer.

ABBREVIATIONS ADH alcohol dehydrogenase BVMOs Baeyer−Villiger monooxygenases BVMOOcean BVMO from Oceanicola batsensis DSM 15984 BVMOParvi BVMO from Parvibaculum lavamentivorans DSM 13023 BVMOAFL838 BVMO from Aspergillus flavus CHMOAcineto cyclohexanone monooxygenase from Acinetobacter calcoaceyicus NCIB 9871 CPOs chloroperoxidases DMSO dimethyl sulfoxide dr diastereomeric ratio FAD flavin adenine dinucleotide FMN flavin mononucleotide FPMOs flavoprotein monooxygenases HAPMO 4-hydroxyacetophenone monooxygenase from Pseudomonas f luorescens ACB HTS high-throughput screening IDO indoleamine 2,3-dioxygenase ISM iterative saturation mutagenesis MD molecular dynamics NAD(P)H reduced form of nicotinamide adenine dinucleotide (phosphate) NMO N-hydroxylating monooxygenase P450 cytochrome P450 P450BM3 P450 from Bacillus megaterium P450cam P450 from P. putida P450pyr P450 from Sphingomonas sp. HXN200 P450SMO P450 from Rhodococcus sp. ECU0066 P450RhF P450 from Rhodococcus sp. NCIMB 9784 P450LaMO P450 from Labrenzia aggregate P450tol P450 from Rhodococcus coprophilus TC-2 PAMO phenylacetone monooxygenase from the thermophilic bacterium Thermobif ida f usca PFCs perfluorinated carboxylic acids PHBH hydroxybenzoate 3-hydroxylase from Pseudomonas species PtlE enzyme from S. avermitilis PenE orthologous protein from Streptomyces exfoliatus PntE orthologous protein from S. arenae pMMO particulate methane monooxygenase PTDH phosphite dehydrogenase RE regioisomeric excess StyAB styrene monooxygenase from pseudomonas sp. strain VLB120 SMOs styrene monooxygenases SidA flavin-dependent monooxygenases Aspergillus f umigatus siderophore sMMO soluble methane monooxygenase TDO tryptophan 2,3-diooxygenase Ty tyrosinase WT wild-type

ASSOCIATED CONTENT Special Issue Paper

This paper is an additional review for Chem. Rev. 2018, 118, issue 5, “Oxygen Reduction and Activation in Catalysis”.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Ning Jiao: 0000-0003-0290-9034 Notes

The authors declare no competing financial interest. Biographies Yujie Liang was born in 1992 in Hainan province, China. He received his B.S. degree at Nankai University (2014). Then he joined Prof. Ning Jiao’s group at Peking University, and he is currently a Ph.D. candidate. His current research interests include (1) developing highly efficient nitrogenation strategies for preparation of nitrogen-containing compounds via inert bond activations and (2) aerobic oxidation as well as oxygenation reactions with economical and environmentally friendly oxidants. Jialiang Wei was born in Hebei province, China. He received his B.Eng. in Macromolecular Materials and Engineering in 2016 from Sun YatSen University. From 2013 to 2016, he was a member of National “TopNotch Undergraduate Program for Pure Science” under the supervision of Prof. Xiaodan Zhao doing research about organoselenium/sulfur catalysis. Currently, he is a Ph.D. candidate in Prof. Ning Jiao’s group in Peking University. His current research interests focus on the design and synthesis of novel and efficient catalysts for aerobic oxidation, halogenation, and amination reactions. Xu Qiu was born in 1995 in Jiang Xi province, China. He received his B.S. degree at Huazhong University of Science and Technology, Wu Han (2017). He is currently a Ph.D. candidate in Prof. Ning Jiao’s group at Peking University. His research focuses on oxygen activation. Ning Jiao received his Ph.D. degree (2004) with Prof. Shengming Ma at Shanghai Institute of Organic Chemistry (SIOC). He then spent 2004−2006 as an Alexander von Humboldt Postdoctoral Fellow with Prof. Manfred T. Reetz at Max Planck Institute für Kohlenforschung. In 2007, he joined the faculty at Peking University as an Associate Professor, and he was promoted to Full Professor in 2010. His current research efforts are focused on (1) developing synthetic methodologies through single electron transfer; (2) aerobic oxidation, oxygenation, nitrogenation, and halogenation reactions; and (3) first-row transition metal catalysis and the inert chemical bonds activation. AA


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