Enzymatic Kinetic Resolution by Addition of Oxygen

Abstract Kinetic resolution using biocatalysis has proven to be an excellent complementary technique to traditional asymmetric catalysis for the production of enantioenriched compounds. Resolution using oxidative enzymes produces valuable oxygenated structures for use in synthetic route development. This Minireview focuses on enzymes which catalyse the insertion of an oxygen atom into the substrate and, in so doing, can achieve oxidative kinetic resolution. The Baeyer–Villiger rearrangement, epoxidation, and hydroxylation are included, and biological advancements in enzyme development, and applications of these key enantioenriched intermediates in natural product synthesis are discussed.


Introduction
Since Pasteur first reported the separation of the enantiomers of tartaric acid via the sodium ammonium and sodium potassium rac-tartrates in 1848, [1] the resolution of racemic starting materials has been an important method for the production of valuable chiral compounds. [2] Advances in asymmetric catalysis have led to many elegant methods for the synthesis of enantioenriched building blocks,b ut kinetic resolution remains akey strategy which is widely used in both academia and industry.
This Minireview aims to complement existing reviews on the resolution of racemic substrates [3] by focusing on recent developments (2010-2020) in enzymatic kinetic resolution involving addition of oxygen. Oxidative kinetic resolution (OKR) processes in which there is no addition of oxygen (for example,t he use of alcohol dehydrogenases/ketoreductases), [4] or in which addition of oxygen occurs as aresult of hydrolysis (such as epoxide hydrolases), [5] and enzymatic desymmetrisation [6] are outside of the scope of this review.W eaim instead to highlight the value of using enzymatic oxidation for the kinetic resolution of racemic substrates as ac omplementary technique to traditional chemical methods.
Thes p 3 -carbon-bound hydroxyl group is arguably the most important and versatile functional group inroad to the assembly of small organic molecules,byvirtue of:1)the possibility of innate stereochemistry at the carbinol centre and welldeveloped stereospecific reactions thereof;2 )oxidation to aketone that links to the vast chemistry of the carbonyl group including,i nter alia, olefination, reductive amination, oxidative ring-expansion, a-alkylation, and 1,2-addition;3 )enabling O-tethered reactions,i ncluding C-H insertion processes;4 )enabling O-directed reactions;a nd 5) acylationa nd sulfonylation chemistry;and so on. As aresult of this central role in synthesis,m ultiple strategies have been devised for accessing enantiomerically enriched or enantiomerically pure (collectively described herein as enantioenriched) oxygenated compounds (Figure 1), including: 1) Enantioselective oxidation. [7] Theu se of an achiral substrate in combination with ac hiral reagent/catalyst to achieve an enantioselective addition of oxygen. 2) Desymmetrisation. [8] As ubset of (1) when the achiral starting material is ap rochiral or meso substrate,w here as ymmetry element in the substrate is removed and chirality is introduced, providing up to 100 %m aximum yield of enantioenriched chiral compounds. 3) a. Classical kinetic resolution (KR). Theselective addition of oxygen to one enantiomer of ar acemic substrate as ar esult of ad ifference in the reaction rates of the Kinetic resolution using biocatalysis has proven to be an excellent complementary technique to traditional asymmetric catalysis for the production of enantioenriched compounds.Resolution using oxidative enzymes produces valuable oxygenated structures for use in synthetic route development. This Minireview focuses on enzymes whichcatalyse the insertion of an oxygen atom into the substrate and, in so doing, can achieve oxidative kinetic resolution. The Baeyer-Villiger rearrangement, epoxidation, and hydroxylation are included, and biological advancements in enzyme development, and applications of these key enantioenriched intermediates in natural product synthesis are discussed. enantiomers;where the enantiomers do not interconvert, the reaction is limited to a5 0% yield of the product. Nevertheless,h igh enantioselectivities of both product and unreacted starting material are possible.S uccessful KR is usually characterised using Chensm ethod of calculating Evalue. [9] b. Divergent reaction on ar acemic mixture (divergent RRM). Thee nantiomers of ar acemic substrate are oxidised by the same reagent producing distinct enantioenriched products.P arallel kinetic resolution (PKR) is as ubset of divergent RRM, where two distinct chiral reagents react with each enantiomer differently to produce two non-enantiomeric products. [10] 4) Dynamic kinetic resolution (DKR). Thesubstrate is able to racemise,e ither spontaneously or via an additional chemical reagent or enzyme,a nd one enantiomer is selectively oxidised. Fora ne ffective DKR the rate of racemisation must be faster than the rate of oxidation of the unwanted enantiomer.Aquantitative yield of product is theoretically possible,t hereby overcoming al imitation of classical kinetic resolution.
Ar ecent resurgence in applications of biocatalysis for asymmetric synthesis [11] has been driven in part by vast improvements in protein engineering techniques,i ncluding but not limited to computation, improved technical protocols, and directed evolution. Analogous to the design and optimisation of chemical catalysts,p rotein engineering has enabled the design of bespoke enzymes to address unique synthetic challenges.For many industrial processes,enzymatic methods may provide attractive alternatives to equivalent chemical reactions as aresult of their generally mild reaction conditions,o ften green and sustainable processes,w ith less hazardous reagents,p roducing less toxic waste and byproducts,a nd reduced costs on large scale. [12] Fore xample, lipase-mediated partial hydrolysis has been al ong-standing and dominant strategy for enzymatic resolution that is widespread in both academic and industrial route development. [13] Enzymes in Nature often evolve to catalyse reactions of specific substrates or specific substrate classes,w hich is advantageous for their application to KR. Within either the wild-type,orvariants bearing specific mutations,anenzymes active site,byproviding an inherently chiral environment, can achieve highly enantiodiscriminating catalysis,r esulting in effective resolutions when presented with racemic substrates. To the enzyme,the substrate enantiomers are totally different molecules whose mirror-image relationship is incidental. Oxygenases,o ften using molecular dioxygen as their [O] source,c atalyse the addition of oxygen to an organic compound either chemo-, regio-, or enantioselectively to yield valuable oxygenated products.Biocatalytic oxidation is becoming accepted as ak ey component in the mainstream organic chemistst oolbox, perhaps still being employed only after classical chemistry has failed, despite such enzymatic methods allowing access to more efficient and elegant syntheses. [14] It is not always viable (practically or economically) to use asymmetric catalysis or other modes of enantioselective synthesis in asynthetic route,and often the racemic synthesis followed by resolution at some point is the superior solution. This Minireview aims to highlight the benefits of using oxidising enzymes for kinetic resolution and stimulate synthetic chemists to include this approach in new syntheses and thereby advance this developing field.
Early reports of enzymatic transformations that involve addition of oxygen were typically of whole-cell microbial processes. [15] Notable examples include a1956 total synthesis of the steroid (+ +)-aldosterone,w here ak ey racemic intermediate 1 was resolved during hydroxylation by the mould Ophiobolus herpotrichus to produce the optically active (+ +)-enantiomer of the oxygenated product 2,l eaving the (À)-enantiomer of the substrate intact (Scheme 1). [16] Furthermore,areport from 1984 described the biohydroxylation of ar acemic 2-azabicyclo[2.2.1]heptane derivative 3 by Beauveria sulfurescens that occurs selectively at the 5-exo CÀHb ond, producing enantioenriched hydroxylactam (+ +)-4. [17]   Thef lavin-dependent Baeyer-Villiger monooxygenase (BVMO) catalyses the oxidation of both cyclic and acyclic ketones to lactones and esters,r espectively. [18] Tr aditional chemical methods to achieve the Baeyer-Villiger (BV) transformation [19] typically employ stoichiometric oxidants containing the peroxy linkage;s uch reagents are often heatand shock-sensitive,toxic, and expensive,all of which severely constrain their large-scale industrial application. [20] BVMOs harness aerial molecular oxygen to produce the reactive oxidant in situ at low concentration, offering as afer,g reen alternative with considerable effort now being made to expand the traditionally limited substrate scope of these enzymes and to improve selectivity. [21] Thecatalytic cycle is initiated with the binding of NADPH and subsequent flavin reduction via hydride transfer from the nicotinamide cofactor (Scheme 2). In contrast to acid-catalysed chemical BV reactions,t he catalytically active species here is the deprotonated peroxyflavin 5.I na ll monooxygenases there is an "uncoupling" decomposition pathway,where non-substrate bound peroxyflavin undergoes proton transfer to produce hydrogen peroxide and regenerate flavin. In BVMOs,N ADP + has been shown to be essential for stabilisation of this deprotonated intermediate which, upon nucleophilic attack onto the carbonyl of the substrate,f orms the key tetrahedral "Criegee intermediate" 6.Itiscrucial for this intermediate to achieve an anti-periplanar alignment of the migrating CÀCbond with the peroxy OÀObond to enable bond migration and, with certain substrates,a lso with an oxygen lone pair. [22] Theregioselectivity is subject to influence by multiple subtle factors;n evertheless,i ti sg enerally predictable,f orming product by migration of the more substituted bond of the substrate.D ehydration of the hydroxylated flavin 7 regenerates flavin 8 to complete the catalytic cycle.
In general, the reaction creates no new stereogenic centres and therefore is only enantioselective through KR of racemic starting materials.D esymmetrisation reactions of achiral precursors ( Figure 2C)a re exceptions to this but fall outside the scope of this review.Such processes can be of the "classical kinetic resolution" type ( Figure 2A)w here one enantiomer is oxidised to the lactone leaving the remaining substrate enantioenriched, or of the "parallel kinetic resolution" type where each enantiomer gives rise to ad ifferent lactone in an enantiodivergent process.Ifthe starting material is able to be racemised, it is possible to achieve aDKR where faster oxidation of one ketone enantiomer can produce up to 100 %yield of enantioenriched lactone ( Figure 2B).

Methodology Overview
Different BVMO subclasses have inherent advantages or disadvantages.F or example,c yclohexanone monooxygenase (CHMO) is widely used because of the large substrate scope  but its reactions can be challenging to scale up due to thermal instability,w hereas phenylacetone monooxygenase (PAMO) is generally more thermally stable but has relatively limited substrate tolerance.Attempts have been made to incorporate the excellent activity of CHMO into PA MO in order to deliver the best characteristics of both prototype enzymes. [23] Cyclohexanones are well-tolerated substrates for avariety of BVMO subclasses,a nd there has been considerable research focused on their transformation to e-lactones.T he Reetz group expanded the substrate scope of PA MO considerably by using directed evolution to identify,from the X-ray crystal structure,t wo distal residues which were able to promote allosteric effects in the shape of the binding pocket of PA MO. [24] This Q93N/P94D variant was shown to catalyse the KR of aw ide range of a-substituted cyclohexanones previously not accepted by WT PA MO (Scheme 3).
BVMO instability has been attributed to unwanted oxidation of sulfur-containing residues caused by H 2 O 2 built up from the decomposition pathway of non-substrate bound BVMO.Reetz described how both the thermal and oxidative stability of aCHMO could be improved by substituting distal amino acid residues Met/Cys for non-sulfur containing amino acids which are less easily oxidised. [25] These new CHMO variants were able to retain up to 40 %a ctivity after incubation with H 2 O 2 .T hese oxidation-resistant mutants showed comparable activity and selectivity compared to WT CHMO for arange of aryl and alkyl substituents (Scheme 3). In ac ontrasting approach, Bornscheuer reported the use of computational methods to develop CHMO mutants with new disulfide bonds that showed improved oxidative stability with no decrease in performance for the KR of 2-methylcyclohexanone. [26] Rudroff reported an in-depth substrate profiling for two new BVMOs:acamphor monooxygenase (CAMO) belonging to the CHMO subgroup and a2 -oxo-D 3 -4,5,5-trimethylcyclopentenyl-acetyl-coenzyme Am onooxygenase (OTE-MO). [27] Unsurprisingly,C AMO showed higher Ev alues for ar ange of cyclohexanones;h owever, both enzymes gave generally poor selectivity with E = 3-69, apart from the substrate with R = Ph which gave E > 200 (Scheme 3). However,b oth CAMO and OTEMO were shown to be excellent catalysts for the desymmetrisation of 3-vinylcyclobutanone and this led to the first chemoenzymatic synthesis of the Taniguchi lactone,akey intermediate in the synthesis of multiple natural products.I narecent report, the Rudroff group was able to use the genomic sequence of ar ecently published and thermostable BVMO,TmCHMO,for database screening and identified an ew BVMO from Amycolaptosis thermoflava. [28] This new thermostable monooxygenase was able to process ar ange of cyclic ketones with excellent conversion and enantioselectivity.
Bornscheuer and Mihovilovic achieved an impressive whole-cell biocatalytic cascade incorporating the BVMOcatalysed KR of racemic 2-methyl cyclohexanone 11 (Scheme 3). [29] Expressing asequence of alcohol dehydrogenase (ADH), enoate reductase (ERED), and BVMO in E. coli allowed the transformation of alcohol 10 through to the corresponding caprolactone 12 in 62 %o verall yield and > 99 % ee. They further developed this cascade by creating af usion protein of the ERED and BVMO,achieving a4 0% increase in lactone production compared to that achieved with the separate enzymes. [30] Enantioenriched methyl-substituted lactones produced enzymatically from CHMO have been used as monomers in lipase-catalysed oligomerisation reactions to form homochiral polyesters with potential applications in material science. [31] Thef ocus of biocatalytic BV KR research has predominantly been on cyclic and bicyclics ubstrates,w ith relatively few reported examples of linear aliphatic ketones as accepted substrates for BVMOs.Inone example using N-protected bamino ketone 14,f our BVMOs tested were all able to produce both the "normal" 15 and "abnormal" 16 esters with excellent Evalues (Scheme 4). [32] After hydrolysis of the ester products with Candida antarctica lipase B( CAL-B), highvalue enantioenriched b-amino acid 17 and b-amino alcohol 18 were obtained. Soon after this initial report, am ore comprehensive study was published using ten BVMOs from varying bacterial origins,i nw hich aw ide range of aryl-and alkyl-substituted ketones were converted, with Ev alues > 200. [33] BVMOs are versatile enzymes whose catalytic activity can be employed to synthesise valuable heteroatom-bearing chiral building blocks by non-BV processes.F or example, they are known to catalyse the oxidation of aw ide range of heteroatoms including nitrogen, selenium, boron, phosphorus,a nd sulfur. [34] Codexis used this reactivity in their largescale synthesis of the chiral sulfoxide Esomeprazole,using an engineered BVMO to circumvent problems associated with the previously used Kagan-Sharpless-Pitchen type oxida-Scheme 3. Scope of mono-a-substituted cyclohexanonesubstrates accepted for arange of reported BVMOs. The reported absolute configurations and ee values for individualp roducts vary between references. Angewandte Chemie tion. [35] There are relatively few examples here of KR as most reports focus on the oxidation of achiral substrates;however, there are reported cases of the OKR of racemic sulfoxides, boronates,and selenides.
A p-hydroxyacetophenonem onooxygenase (HAPMO) was reported to catalyse the oxidation of racemic sulfoxides to sulfones in an enantioselective manner,l eaving enantioenriched unreacted sulfoxides. [36] Alkyl substituents were found to be better tolerated than aryl;h owever, high conversions and short reaction times (0.5-5.0 h) were achieved with all substrates (Scheme 5). Bornscheuer identified eight new monooxygenases from the eukaryote Yarrowia lipolytica, two of which were able to accept both sulfides and sulfoxides as substrates. [37] Thea uthors reported high enantioselectivities for the asymmetric oxidation of sulfides (methyl phenyl sulfide (MPS), methyl p-tolyl sulfide (MTS), and l-methionine) to the corresponding sulfoxides with multiple variants. Further efficient oxidation to the corresponding sulfones was possible;h owever, there was poor discrimination between sulfoxide enantiomers resulting in alow degree of KR.
Despite poor reported Ev alues for the oxidation of phenylethylboronates,c atalysed by either PA MO [38] or its M446G mutant, [39] this transformation produces two valuable products:t he secondary alcohol and the unprocessed boronate,both with high optical purity.

Modes of Kinetic Resolution
As illustrated previously (Figure 2), KR can proceed via enantioselective and enantiodivergent pathways or, if racemisation of the substrate is possible,v ia DKR.
Gotor and Fraaije took advantage of acidic substrates to achieve DKR. Thecommonly used M446G mutant of PA MO produced ar ange of 3-alkyl-3,4-dihydroisocoumarinsw ith high enantioselectivities (Scheme 6a), although the remaining a-alkyl indanones substrates showed low ee (all < 35 %). [41] This class of substrates readily racemise under the mildly basic reaction conditions (pH 8-10) and it was Scheme 4. a) Initial report and b) further expansiono fthe scope of tolerated N-protected b-amino ketones to the corresponding "normal" or "abnormal"e sters. TenB VMOs from differing bacterial origins were tested (CHMO Acineto ,CHMO Arthro ,CHMO Brachy ,C HMO Xantho ,C HMO Rhodo1 , CHMO Rhodo2 ,C HMO Brevil ,C DMO, HAPMO ACB ,and PAMO) with the highest Evalue reported here;s ee ref. [33] for full reported Evalues for each BVMO. Where the Enumber is reported only as E > 200, either one or all the BVMOs with reported activity gave E > 200.

Angewandte
Chemie found that the addition of small amounts of organic solvent (MeOH or hexane) improved enzyme activity or selectivity. Thea uthors extended this methodology using two linearketone-converting BVMOs,P AMO and HAPMO,toconvert ar ange of a-substituted b-ketoesters,w hich spontaneously racemise at pH 9, to the corresponding diesters (Scheme 6b). These products are easily hydrolysed via chemical methods to produce valuable enantioenriched a-hydroxy esters. [42] Thea uthors intended to extend this concept to include acyclicbenzyl ketones;however, for this class of compounds, the addition of an anion-exchange resin was required to promote racemisation. [43] It was found that less basic resins achieved substrate racemisation without adversely affecting the activity of the isolated HAPMO,a lbeit with prolonged reaction times (selected examples,S cheme 6c). Replicating previously successful conditions,u sing the mutant M446G PA MO and as mall amount of hydrophilic solvent, the same authors were able to further improve the performance of both the standard and dynamic kinetic resolution for these substrates (Scheme 6d). [44] Thea ctivity of BVMOs is generally inhibited by ahigh concentration of substrate and product but Bornscheuer and co-workers were able to overcome this issue by using ahydrophobic adsorbent resin. [45] This allowed both the slow release of substrate and the adsorption of product, such that the HAPMO-catalysed KR of these substrates proceeded with Ev alues > 100 on as ynthetically useful scale (5 mmol).
Asignificant disadvantage for BVMO biotransformations compared to chemical methods of oxidation is the requirement for stoichiometric amounts of the expensive cofactor NADPH and for large scale applications it is essential to develop effective nicotinamide cofactor regeneration sys-tems. [46] Parallel interconnected kinetic asymmetric transformations (PIKAT) allow an ADH to regenerate NADPH through the selective oxidation of secondary alcohol 19, leaving the unreacted enantiomer of alcohol 19 with high optical purity. [47] TheB VMO-mediated kinetic resolution of ketone 20 shown in Scheme 7occurs with an excellent Evalue (E > 200) leading to the concurrent production of three valuable chiral compounds.

Targeted Applications
OKR using BVMOs has many advantages in complex target synthesis,both for natural product synthesis and for the production of other challenging compound classes.T urner and Procter used aC HMO from Acinetobacter calcoaceticus to catalyse the KR of awide range of five-and six-membered cyclic ketones to lactones with excellent Ev alues (Scheme 8a). [48] This was the first report of aB VMO kinetic resolution of a,a-dialkyl cyclic ketones,with previous work in this area focusing on a-monoalkyl cyclic ketones (cf. Scheme 3). Thea uthors develop the methodology further in an enantiodivergent process (Scheme 8b); in this,b oth lactone and remaining ketone proved to be suitable substrates for aS mI 2 -mediated reductive cyclisation, producing enantioenriched bridged or fused cycloalkanols which feature as key core structures in ar ange of complex natural products (examples in Scheme 8c).
TheM ihovilovic group obtained naturally occurring fragrance lactones by whole-cell BVMO biotransformations on ap reparative scale (100-150 mg). [49] In this report excellent Ev alues were achieved in both analytical-and preparative-scale reactions of the six-membered-ring jasmine lactones and their caprolactone homologues (Scheme 9). This was extended by combining continuous-flow hydrogenation with ac yclododecanone monooxygenase catalysed Baeyer-Villiger oxidation (BVOx) in as ingle-operation chemoenzymatic sequence to produce Aerangis lactones. [50] Thek inetic resolution of cis-21 occurred with excellent selectivity (E > 200) and is the first reported case of ad iastereoselective BVOx to produce the natural diastereomer of an Aerangis lactone as the only product (> 99 %d e).

Angewandte
Chemie ate, [52] Mihovilovic reported ar egiodivergent cyclopentadecanone monooxygenase (CPDMO)-catalysed oxidation producing the lactone 25,w hich leads to the non-natural enantiomers of these natural products. [53] Fused bicyclic ring systems are excellent substrates for BVMOs due partially to the release of ring strain, and there are many examples of the oxidation of racemic fused cyclobutanones. [54] Thec yclic ketone 26 (Scheme 11) is an exceptional model system to study BVMO OKR because its structural features lead to interesting and diverse products through the enantio-and regioselective oxidative transformation. Through genome mining,O pperman found four phylogenetically distinct new BVMOs from the fungus Aspergillus flavus,t hree of which were able to catalyse the oxidation of ketone 26 in up to 99 % ee. [55] Rial also used genome mining to identify seven new BVMOs,t wo of which were able to provide the abnormal lactones (AL) selectively from the model bicyclic ketone 26 (Scheme 11 a). [56] Tw oo f the new BVMOs gave an effective KR, each yielding one enantiomer of the abnormal lactone selectively with E > 200. This is the first report of ap rokaryotic BVMO giving the abnormal (+ +)-lactone selectively.

Epoxidation
Epoxidation by chemical means has long been established as an effective method for the KR of racemic olefin substrates, [74] including pioneering work by Sharpless, [75] Jacobsen, [76] Katsuki, [77] and Shi. [78] Although there has been considerable research into biocatalytic asymmetric epoxidations, [79] there are relatively few examples that extend this methodology to achieve KR. Styrene monooxygenases are commonly used biocatalysts for this transformation. Similar to BVMO they are flavoproteins,b ut consist of two components.T he reductase StyB uses NADPH to catalyse the reduction of flavin 8 to FADH 2 9,which is then transferred to the oxidase StyA to undergo ar eaction with molecular oxygen to produce the peroxy-flavin 5H + as the active epoxidising agent (Scheme 12). Thes ystem is often paired with aregeneration system to re-form NADPH from NADP + .
Early literature on styrene monooxygenases (SMOs) focused solely on aromatic sulfides and alkenes,s uch as styrene derivatives;h owever,i n2 010 Wu reported the first asymmetric epoxidation of nonconjugated alkenes by the novel SMO StyAB2, [80] thereby opening up the potential for am uch wider substrate scope. [81] It was found that the presence of ah ydroxyl group in the substrate significantly improved the ee (Scheme 13 a) and enabled application to the kinetic resolution of allylic secondary alcohols (Scheme 13 b). [82] Tw on ew bacterial SMOs,P aSMO and MlSMO, were identified through genome mining of Paraglaciecola agarilytica and Marinobacterium litorale,r espectively.T hese enzymes showed improved activity and selectivity in whole-cell biotransformations for asymmetric epoxidation;however, no Evalues were reported for the allylic alcohol tested. [83] Using ar econstructed ancestral protein from six known SMO enzyme sequences,C hen used genome mining to identify anew monooxygenase from Herbaspirillum huttiense (HhMo) able to catalyse the epoxidation of nonconjugated terminal alkenes. [84] Excellent enantioselectivities were achieved (> 99 % ee for all examples) and far superior results were obtained by comparison with traditional chemical methods such as the Sharpless epoxidation (Scheme 13 c).

Angewandte Chemie
Importantly,t his work is the first to report tolerance of aliphatic substrates in am onooxygenase-catalysed epoxidation.

Kinetic Resolution via C-H activation
Direct functionalisation of unactivated C À Hb onds has emerged as one of the most important focuses in modern synthetic organic chemistry, [85] with many elegant applications to KR. [86] Among the many advantages in employing enzymes in synthesis is the ability to functionalise positions in ac ompound which are inaccessible or inactive towards chemical reagents;t his ability has been employed in many chemoenzymatic total synthesis applications.D espite this, enzymes have been largely overlooked as reagents to achieve the deliberate resolution of racemic intermediates via C-H activation;f requently,s tudies only utilised the native substrate in conjunction with the known corresponding enzyme from its biosynthetic pathway.
One of the most important enzyme classes for oxidation via C-H activation is the hemoprotein cytochrome P450s. [87] Thec atalytic cycle initiates with substrate binding and displacement of aw ater molecule,i nducing an active site conformational change (Scheme 14). Electron transfer from the associated P450 reductase domain produces an Fe II species 30 which reacts with molecular oxygen to produce 31 which, after another electron transfer, forms the ferric peroxy complex 32.T wo subsequent protonations,along with al oss of water and heterolytic cleavage of the OÀOb ond, results in the formation of the highly active ferryl Compound Is pecies 33,w hich is the key intermediate for substrate oxidation. Release of oxidised substrate and binding of aw ater molecule completes the catalytic cycle.
Hertweck and De Paolis reported the first parallel kinetic resolution using the P450 monooxygenase AurH in their synthesis of aureothin (34), [88] expanding on Hertwecks earlier report of the first chemoenzymatic total synthesis of this natural product. [89] This unprecedented regioselective C À Hb ond oxidation, and subsequent cyclisation, installed the tetrahydrofuran ring in the final step,t hereby circumventing issues such as olefin isomerisation and stereocentre epimerisation that had complicated previously reported syntheses. [90] In this transformation, the natural product is produced through selective allylic hydroxylation of one enantiomer and cyclisation with both high yield and enantioselectivity, recovery of some enantioenriched starting material (19 % yield, 85 % ee), and production of the unexpected racemic pyran 35 (Scheme 15). Thepyran is proposed to arise through oxidation at a-o rb-p ositions (highlighted in green) of the other enantiomer,then elimination, tautomerisation, and 6p- Lütz reported aK Rw hile investigating the enantioselective whole-cell oxidation of b-ionone (37)u sing P450 BM3 enzymes. [91] Am oderate ee was established for (R)-4hydroxy-b-ionone 38 produced within short reaction times; loss of enantiomeric purity occurred in prolonged reactions due to over-oxidation to the corresponding ketone (Scheme 16 a). This suggests that the (R)-hydroxylated product 38 is the preferred enantiomer in the oxidation to 4-oxo-bionone,a nd this was confirmed in an experiment with the oxidation of the racemic alcohol 39 as starting material which returned enantioenriched (S)-alcohol 40 (Scheme 16 b). The reported Ev alue is low (E = 2) but the transformation represents an important proof of principle that P450 monooxygenases are capable of achieving KR of racemic alcohols.
Robertson and Wong reported the kinetic resolution, by P450 BM3 mutants,o fi ntermediates in the formal synthesis of eleutherobin (44)a nd oxidised analogues. [93] As part of this study,l actone 45 was screened against ap anel of 24 mutants to give adiverse set of hydroxylated products (Scheme 17). In as mall-scale preparative reaction (42 mmol), using mutant RT2/IP,a lcohol 46 was obtained in 64 % ee and 36 %y ield.
Soon after the publication of ar eport on the role of a2oxoglutarate-dependent dioxygenase (2-ODD) in ak ey step of the biosynthetic pathway of etoposide (47), [94] two groups described chemoenzymatic routes to podophyllotoxin (49) patterned on this biosynthetic step.I nt he first, Kroutil and Fuchs reported an impressive large-scale enzymatic kinetic resolution of rac-48 via enantioselective ring-closing CÀC bond formation. [95] Although this transformation was shown not to proceed through ah ydroxylated intermediate,t he authors reported initial results in which one substrate enantiomer was selectively hydroxylated to 50,l eaving enantioenriched starting material behind. Thes tereochemistry at the benzylic alcohol centre in the substrate determines the reactivity (Scheme 18), leading to the proposal that substrate positioning within the 2-ODD-PH active site is crucial. In the second report, Renatasg roup used enantio-

Angewandte
Chemie pure starting material 48 in agram-scale oxidative cyclisation, achieving excellent yields of 51 and installing the hydroxy functionality at alater stage. [96] Both groups prepared arange of podophyllotoxin-related structures to test the 2-ODD-PH substrate scope and reported ar ange of both hydroxylated and cyclised products.

Summary and Outlook
This Minireview highlights the considerable research focus on BVMOs,a nd the body of literature is much larger than for other biocatalytic oxidative kinetic resolution transformations such as epoxidation or hydroxylation via C-H activation. Even so,there remains,ingeneral, alack of direct application of the results to synthesis,w hether for natural product chemistry or use in industrial routes to,for example, pharmaceuticals.B iotechnologists have focused on enzyme development, leading to impressive advancements in enzyme stability and scalability,aswell as discovering transformations that produce interesting molecules in enantioenriched form with synthetically useful conversions and high selectivity. Many of these molecules have been employed by synthetic chemists in routes to natural product intermediates or privileged structure classes;m ore often than not, however, the two worlds have not intersected, with the chiral intermediates actually taken on in multistep synthesis not necessarily being sourced by biocatalytic oxidative KR.
There is,h owever, ag rowing number of academic and industrial groups who conduct research at the interdisciplinary interface of biocatalysis and chemical synthesis,i n addition to those willing to collaborate,allowing the transfer of knowledge and skills.T here are huge opportunities for methodological and technological advances in biocatalysis to influence organic synthesis profoundly, [97] and vice versa. We are confident that the field will continue to flourish with the development of elegant new strategies and methods arising from seamless applications of both chemical and enzymatic transformations in complex target syntheses. [98]