Applications of Oxygenases in the Chemoenzymatic Total Synthesis of

Nov 15, 2017 - Nature has produced a diverse range of oxygenases for the modification of secondary metabolites with selectivity profiles that are unma...
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Perspective

Applications of Oxygenases in the Chemoenzymatic Total Synthesis of Complex Natural Products Emma King-Smith, Christian R Zwick, and Hans Renata Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00998 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 16, 2017

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Biochemistry

Applications of Oxygenases in the Chemoenzymatic Total Synthesis of Complex Natural Products Emma King-Smith,‡ Christian R. Zwick III,‡ Hans Renata* Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458

Supporting Information Placeholder ABSTRACT: Nature has evolved a diverse range of oxygenases for the modification of secondary metabolites with selectivity profiles that are unmatched by conventional man-made catalysts. In the past two decades, organic chemists have begun to harness the synthetic potential of these biocatalysts to develop efficient chemoenzymatic synthesis of complex natural products. Judicious combination of synthetic and enzymatic transformations in multi-step synthesis can often result in powerful disconnections that compare favorably with contemporary chemical strategies to access the target natural products, while at the same time, presenting opportunities to innovate. This Perspective highlights strategic applications of enzymatic hydroxylation to simplify problems in natural product synthesis. Finally, newly discovered enzymes that would facilitate further developments in this field are discussed.

Introduction Since the advent of medicine, mankind has relied on natural compounds from animals, plants, and microbes as a source of therapeutic agents to treat diseases. In the past century, advances in various fields of chemistry have allowed the isolation of active principles of such natural extracts and the determination of their molecular structures and modes of action. This discovery in turn spurred the development of the field of natural product synthesis which was driven by both the biological promise and the fascinating architecture of complex natural products. To date, natural products remain fertile ground for drug discovery. A recent survey by the US Food and Drug Administration (FDA) indicated that natural products and their derivatives still account for more than 30% of approved small molecule drugs in the last three decades.1 This notion was further bolstered by recent FDA approvals of natural product-derived drugs such as Adcetris and carfilzomib for cancer treatment.2 In addition, we continue to rely on natural antibiotics such as vancomycin and tetracycline to treat microbial infections. While the complex three-dimensional architectures of natural products are often credited for their ability to bind specific receptors, they also pose tremendous problems for de novo preparation. As a result, despite rapid developments in organic methodology, it remains challenging to devise efficient synthetic strategies that allow scalable and practical production of some of these compounds for further biological testing and medicinal chemistry optimization. This is especially true for natural products possessing highly congested ring structures and/or rich array of functionalities. Thus, many natural product syntheses often require long synthetic sequences that involve extensive protecting group manipulations and functional group interconversions.3 Similarly,

even though methods of metabolic engineering have recently gained traction for heterologous natural product production (e.g. taxol), biological systems are highly complex and not trivial to manipulate.4 Natural product biosynthetic machineries arise from millions of years of natural evolution and developing designer microbial cell factories from these pathways for large-scale production of secondary metabolites and related analogs can be an arduous task. This work typically entails extensive optimizations, not only to improve the catalytic activities of individual enzymatic steps, but also to allow precise regulation of metabolic fluxes and ensure that various pathways work in tandem to afford high titers of target compounds. In recent years, efforts to access natural products have begun to explore the possibility of combining the tools of organic synthesis and biosynthetic enzymology for efficient chemoenzymatic synthesis.5 Such an approach has the distinct advantage of being able to leverage the discovery of novel enzymes with unique reactivity and functional group compatibility to solve challenging chemo- and stereoselectivity issues in organic chemistry, while at the same time, relying on modern advances in chemical synthesis for efficient building block preparation or further downstream manipulations. When carefully designed, chemoenzymatic synthesis can allow efficient access to therapeuticallyrelevant natural products. In this Perspective, we present case studies of successful integration of enzymatic reactions in total synthesis campaigns, focusing on the strategic applications of enzymatic hydroxylation. The select case studies are organized according to enzyme families and each section begins with a brief primer on the structural and catalytic features of the enzyme family in context. The transformative power of these enzymes is next illustrated through several case studies that highlight creative chemoenzymatic approaches to natural product synthesis. Finally, we end by describing a few untapped discoveries that might merit further attention for application in chemical synthesis. Cytochrome P450s Among the various families of iron-dependent oxygenases, cytochromes P450s have been the most extensively studied from both mechanistic enzymology and biotechnological application standpoint. Capable of a diverse array of oxidative transformations,6a this enzyme family is characterized by the presence of a heme cofactor and axial ligation of a Cys residue to the iron center, which in turn tunes the electronics of the iron center for reductive activation of molecular oxygen.6b This activation pathway eventually leads to the formation of a reactive oxoiron(IV)porphyrin π cation radical (“compound I”, Figure 1), which in turn abstracts a hydrogen atom from the substrate. In the case of small molecule hydroxylation, a subsequent “radical rebound” event leads to recombination of the substrate radical and Fe-bound

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OH• to generate the hydroxylated product. One of the key structural features of the P450s is the requirement of a redox partner that will allow subsequent deliveries of electrons to the heme center to generate the reactive compound I species. Biochemical studies have shown that the majority of P450s in nature exist as multiprotein systems, consisting of standalone heme and reductase proteins.7 However, a few P450s, such as P450BM38 and P450RhF,9 are fused to their reductase partners, and due to their self-sufficiency, are highly appealing for use as practical biocatalysts. In recent years this family of enzymes has seen tremendous development, primarily in the areas of enzyme engineering and synthetic chemistry.10 Although the list of examples is brief, these enzymes have also seen application in the synthesis of biologically relevant secondary metabolites. In this section, we wish to highlight a few of these examples in order to demonstrate the power of late-stage P450 catalyzed C–H hydroxylation in natural product total synthesis. OH 2 N

OH

N Fe III

R N

N

N

A

R

N

N

N

Me

S

N

N

N N Fe N N

Fe N

Me

N

S

S

R N

HO 2C

H

O

N N

S compound II

A

N

Cys CO2H

R

N N

R N

O

N

Fe IV N

HO N

N

S

O

R

N H 2O

H+

OMe

Me 9aMe

O

Me

Me

7

I Me

O

O

9a

O

50% over 2 steps

CO2Me Me

9a

Me

Me

8

h) Sn2Me 6, Pd(PPh 3)4; 9, CuI, Pd 2dba3, reflux, 85%

Me

O

Me

Me

7

OMe

O2N

S

Me

O

O

9a

O

CHO

N S

Me

Me I

OMe

O

B Me

Me O

Me 9a Me

I CO2Et O2N

Me 5

3

Ar

The polyketide secondary metabolite aureothin (1) has been extensively studied due to its interesting structure as well as anti-tumor, antifungal, and pesticidal activities. Although aureothin was first characterized in 1961 (isolated from Streptomyces thioluteus), the past 10–15 years have seen an explosion of publications (>100) studying or referencing this natural product.11 Notably, racemic total syntheses by Baldwin, Trauner, and De Paolis have been reported,12 but asymmetric total synthesis remains elusive due to facile racemization of the substituted furan moiety. The biosynthesis of aureothin involves a combination of polyketide synthase (PKS) and shikimic acid pathways.13 In 2004, the Hertweck group identified the presence of a pyrone methyltransferase AurI and a multifunctional cytochrome P450 monooxygenase AurH in aureothin biosynthetic pathway.13d AurI is responsible for the O-methylation of aureothin’s pyrone ring, while AurH assembles aureothin’s furan motif through a C–H hydroxylation/C–H etherification cascade. As this biocatalytic cascade occurs late in the biogenesis of aureothin, its use in chemoenzymatic total synthesis could offer an elegant solution for the enantioselective synthesis of aureothin. This idea was put into practice by the Hertweck group in a total synthesis of

O

(+)-aureothin (1)

H +, e–

(+)-Aureothin

25% over 2 steps

6

P450 oxidation

N

Figure 1. The P450 catalytic cycle featuring hydrogen abstraction by compound I, followed by radical rebound for substrate hydroxylation.

Me

7 g) AurH (S. lividans ZX1 whole cells), 32–75%

Fe III N

N

Fe III

compound I

H

9a

e) DBU, 80 ºC Me f) MeOSO F 2

O

H

N

Me

4

I

S

N

I

I

N

R

c) 5, NaH, nBuLi CHO d) DMP

42% over 2 steps

N

H N

CO2Et Me 2 O

Fe II

O

a) 3, KHMDS b) DIBAL

Me

N

O

O

H

O2

B, C

S

N

e–

Me

O

Ph N

S

Me

Features: A. Substrate binding induces spin shift, allowing Fe(III) to Fe(II) reduction B. Cys ligation increases basicity for H abstraction C. Radical rebound mechanism for hydroxylation

Fe IV N

N Fe III

N

(+)-aureothin that combines chemical construction of its carbon skeleton with AurH-catalyzed oxidation and cyclization (Scheme 1A).14a This synthesis commenced with a modified Julia olefination, reduction, aldol, and oxidation sequence, affording tricarbonyl compound 6 in 11% yield over 4-steps. Next, condensation and methylation afforded pyrone 7 in a 50% two-step yield. Notably, methylation could be performed chemically utilizing methylfluorosulfonate in excellent yield (80%). In order to perform the oxidative cascade, AurH was heterologously produced in S. lividans ZX1. To avoid challenges associated with electron transport and cofactor regeneration, the enzymatic transformation was performed in whole cells, affording the furan product in 32–75% yield. A final Stille coupling resulted in (+)-aureothin in 85% yield (4% overall yield over 8-steps). It is worth noting that Hertweck and De Paolis have developed a second generation chemoenzymatic synthesis of 1 (Scheme 1B) featuring an AurH-mediated oxidative kinetic resolution of a substrate lacking oxidation at C9a carbon (10).14b Scheme 1. Chemoenzymatic synthesis of (+)-aureothin utilizing late-stage biocatalytic oxidation cascade with AurH.

H

N

S

Fe III

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10

OH

9

AurH (whole cells)

Ar OMe 42% yield 99% ee Ar = 4-NO2Ph

O

Me

Me

7

Me

9a

O

O

OMe

1

Tylactone and Juvenimicins Erythromycin, pikromycin, and tylosin are part of a closely related family of 14 and 16-membered macrolactone glycosides.15 They display various activities from immunosuppressant to antitumor, but are most noteworthy due to their antibiotic activities.16 This family of related natural products shares a similar mechanism of action: they bind the 50S ribosomal subunit adjacent to entrance of the peptide exit tunnel. Biosynthetically, they are constructed using three major groups of enzymes: polyketide synthases (PKSs), glycosyltransferases, and cytochrome P450 monooxygenases.17 Unfortunately, due to the current antibiotic crisis, these natural products are becoming less effective. Therefore, the development of structurally novel macrolides is of dire importance. Towards this aim, the Sherman group has recently reported an efficient strategy towards late-stage diversification of tylactone, the biosynthetic precursor to tylosin (Scheme 2).18 A hexaketide intermediate 11 was first constructed via various aldol, enolate, and Horner-Wadsworth-Emmons transformations. Subsequently, 11 was

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Biochemistry

loaded to the ACP domain of JuvEIV via trans-thioetherification to initiate in vitro catalysis with JuvEIV and JuvEV modules, which 1) install the final four carbons of the macrocycle 2) introduce three chiral centers and 3) perform the final macrocyclization step, producing tylactone in 69% yield. Previously described mutant S. venezuelae strain DHS316, lacking the pikromycin PKS and P450 genes but containing the des glycosyltransferase genes, was next utilized to synthesize macrolide M-4365 G1 (12) in 70% yield, setting the stage for the key oxidative diversification using various P450s belonging to the biosynthesis pathways of related macrolides. By varying the order of P450 reactions, various juvenimicin and M-4365 analogs could be accessed in an expedient manner. C20 and C23 hydroxylation were performed utilizing P450 enzymes TyII and MycCI, respectively, while C12–13 double bond epoxidation was performed by JuvD. The chemoenzymatic total syntheses of juvenimicin B1, B3, A3, and A4 were completed in 16–18 linear steps through this strategy.

tion, Tsuji-Wacker oxidation, and Robinson annulation generated ketone 19 in 73% over 3-steps.21 Bromination of 19, Suzuki coupling, and condensation then afforded protonated imine 21 in 55% yield over 3-steps. At this stage, several known C—H oxidation conditions were tested, but typically resulted in poor positional selectivity, generated inseparable mixtures, or resulted in over-oxidation. The solution came from a screening campaign of various P450BM3 mutants which have previously been evolved for selective oxidative deprotection of glycosides.22 Through an LC-MS-based screening, variant 8C7 was found to provide oxidation at the desired carbon with greater than 2.5:1 regioselectivity. This biotransformation was performed on 160 mg scale with concomitant use of a NADPH regeneration system consisting of an alcohol dehydrogenase and 2-propanol to prevent the stoichiometric use of this expensive electron donor. Finally, chemical oxidation with DMP provided nigelladine A (16) in 21% yield over 2 steps (43% brsm).

Scheme 2. Chemoenzymatic total synthesis of the juvenimicins using late-stage oxidative diversification of M4365 G 1 .

Scheme 3. Chemoenzymatic total synthesis of nigelladine A via regioselective allylic oxidation catalyzed by an engineered P450 enzyme. O

JuvEIV KR O

O

OH

KS

Et

Me

Me

11

i-Pr

JuvEV

Me

KS O Me Me Me O

Et

KR

OH

O

k) P450 8C7, NADP, ADH, 2.5% DMSO, 0.8% iPrOH, kPi (pH 8), 23°C Me

Me

O

Me

23

Et

20

13 Me O

O HO

O

Me

TylI

Me

12

O

Me 61% NMe 2

OH

Me

TylI, MycCI

Et

O

32% over 2 steps

oxidative diversification with P450s

63%

JuvD

OH 22

O CF3

55 % over 3-steps

Me

Me

Me

O nigelladine A (16)

CF3

Me Me NHBoc

N

O BPin

21% over 2 steps i-Pr (43% brsm)

Me i-Pr 19 (87% ee)

Me l) DMP, DCM

O

20

(4-CF3-C6H 4)2P

N

tBu (R)-CF3-tBuPHOX

XPhos Pd G2 : 2nd gen. Buchwald XPhos precatalyst ADH : alcohol dehydrogenase

Rieske Oxygenases

Me OH

O

Me NMe 2

O

Me HO

Et

O

OH

Me

Me O

O HO

i-Pr

h) NBS, MeCN i) 20, XPhos Pd G2, j) TFA, DCM; NaHCO 3; SiO2

Me

NH

73% yield over 3 steps

juvenimicin B1 (13)

O

23

20

13 Me O

M-4365 G1 (12)

Me

OH 12

g) KOH, xylenes, 110°C 18

21

Me

N

Me

O Me

Me

O

Me i-Pr

NMe 2

e) Pd 2(dba) 3, (R)-CF3-tBuPHOX f) PdCl 2, CuCl

O

i-Pr

O

P450-catalyzed allylic hydroxylation

Me

M-4365 G1 (12)

Me

c) 1M HCl d) MeI, NaH 66% over 4 steps

Me

AT ACP TE

48% overall

O HO

O

17

then DHS316 cells Et

Me

Me

AT ACP

Me

PhS

O

a) Me 2NNH 2, TFA b) LDA, allylchloroformate

Me

20

O HO OH

juvenimicin B 3 (14)

O

Me NMe 2

Me Et

OH

O

12 13 Me

O

O

O HO OH

O

Me NMe 2

juvenimicin A4 (15)

Nigelladine A In 2014, Chen et al. isolated three norditerpenoid alkaloids, nigelladines A–C, from the Nigella glandulifera plant.19 Each alkaloid possessed novel carbon skeletons containing a highly conjugated π system and displayed protein tyrosine phosphatase 1B inhibitory activity without cytotoxicity in A431 cells. In order to evaluate the viability of latestage C—H oxidation strategies, a synthesis of nigelladine A (16) was pursued in a collaborative effort between the Arnold and Stoltz groups (Scheme 3).20 The synthesis began from known enone 17, which was constructed through a Stork-Danheiser transposition from 1,3cyclohexanedione. Next, β ketoester 18 was synthesized through a 4step sequence comprising hydrazine formation, acylation, hydrolysis, and methylation in 66% yield. Subsequently, asymmetric allylic alkyla-

Rieske oxygenases are a family of multi-component enzymes that are capable of activating molecular oxygen and possess a signature Riesketype [2Fe-2S] cluster coordinated by two cysteine and two histidine residues.23 This domain is responsible for the transfer of electrons to the non-heme iron center where oxygen reduction and substrate oxidation occur. Rieske oxygenases were first identified in bacteria capable of degrading aromatic compounds to the corresponding cisdihydroxylated metabolites.24 Subsequent work eventually characterized these enzymes as naphthalene dioxygenase and toluene dioxygenase. Though the catalytic cycle of these enzymes still needs to be fully elucidated, mechanistic evidence suggests the formation of a hydroperoxide complex which undergoes a O–O cleavage and forms an Fe(IV)=O species upon reaction with the aromatic substrate (Figure 2).25 An alternative pathway consisting of a discrete O–O cleavage followed by formation of Fe(V)=O(OH) species prior to substrate oxidation has also been put forth. The two proposed pathways eventually lead to the formation of a ferric alkoxide complex, which upon reduction and protonation leads to product release and regeneration of the non-heme iron center. Since its discovery by Gibson,24 toluene dioxygenases (TDOs) from Pseudomonas putida have seen extensive use in chemoenzymatic total synthesis. The enantioselective dearomatization and desymmetrization have been utilized in the syntheses of many natural products such as

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(+)-lycoricidine,26 ent-deoxydihydrotsugicoline,27 and xylosmin.28 The wide range of natural products that can be formed from cis-1,2dihydroxyarenes arises from a few select features (Figure 2). Firstly, the diols have a pro-enantiotopic plane of symmetry which facilitates diastereoselective functionalization of the alkenes.29 The ring substituents provide additional functional handles for further manipulations. For example, halogen substituent such as a bromine or chlorine lends itself as a partner in cross coupling reactions. The diene moiety is also capable of undergoing cycloadditions or other selective olefin functionalizations, including epoxidation, aziridination and oxidative cleavage.30 Finally, the opposite stereochemistry of the dihydroxylated product can be obtained by using dioxygenase from Ralstonia eutropha B9. This section highlights recent aplications of TDO-catalyzed dihydroxylation in the synthesis of complex natural products.

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olefination of the highly hindered carbonyl provided olefin 27. Protecting group manipulations afforded alcohol 28, which was oxidized to the corresponding ketone using a Ley-Griffith oxidation.34 Addition of isopropenyllithium led to the formation of 30, which underwent an anionic oxy-Cope rearrangement upon treatment with sodium hydride. A reductive cyclization reaction with samarium(II) iodide gave alcohol 32 and established the tricyclic core of patchoulenone. Debenzylation, Parikh–Doering oxidation, and dehydration completed the synthesis of the target natural product. Scheme 4. Chemoenzymatic synthesis of (–)patchoulenone via stereoselective arene dihydroxylation with TDO. Me

OH 2

R His OH

O

O2, H +, e-

His

OH

O 24 (from TDO dihydroxylation)

Asp

O

(non-heme Fe center)

Cl

Cys Cys

O

Fe

H +, e-

S

S

His

Fe

OH

His His

O His His

His

R

OH

R

Li

25

O

i) TPAP, NMO

Me

BnO

Fe III 30

O His

Asp

His

OH

R O

O

His

O

OH

His

Asp

Me

Me

His

OH

Me

O

27

OBn

Me

Me Me Me

Me HO

31

Me

g) BnBr, NaH h) DDQ

Me

O

Me

O

m) H 2, Pd/C Me n) SO 3•pyridine, Et 3N

l) SmI2

Asp O

Me

Me

Me

O

His

Me 28

R

Fe V

BnO

Cl

O

f) DIBAL

Me

76% over 6 steps

k) NaH

Fe IV

Ar

HO

Me

Me

CN 26B 80% b) KOH c) MeI, NaHMDS d) H 2, PtO2 e) Ph 2PMe 2I, nBuLi; tBuOH

Me

j) 29

Me

O O

+

29

HO

O

O

O

Cl 26A 20%

Me

Asp

CN

CN

Fe III

(Rieske center)

Me

quant. yield

H

Ar

O

O

Ar

O

Fe II

H

Ar a) 25

o) SOCl2, pyr, DMSO

OBn

34% over 4 steps

32

Me O (–)-patchoulenone (23)

R

Scheme 5. Chemoenzymatic total synthesis of ent hydromorphone via stereoselective arene dihydroxylation.

Fe IV O O Asp

X

cross coupling

E+ Br

OH Diels–Alder OH epoxidation, ozonolysis, aziridination

X OH OH

pro-enantiotopic plane

E+

H

H

10-15 g/L TDO dihydroxylation

34

b) AcOH,

Br

a) E. coli JM109 (pDTG601A)

KO 2C

OH

HO

35

(–)-Patchoulenone

50%

Though various modes of cycloadditions should be possible, synthetic manipulations of the diene moiety of TDO products have focused primarily on the Diels-Alder cycloaddition. Application of this strategy can be found in the synthesis of (–)-patchoulenone (23) by Banwell (Scheme 4).31 First isolated in 1964 from Cyperus rotundus Linné, this compound shows potent anti-fungal activity against Rhizoctonia solani and Saprolegnia asterophora, as well as in vitro activity against malarial parasite Plasmodium falciparum.32 The sequence started with the protection of microbially oxidized toluene (reported up to 15 g per liter)33 with p-methoxybenzaldehyde dimethyl acetal and Diels-Alder cycloaddition to give a 4:1 mixture of 26A:26B in quantitative yield. Hydrolysis of both diasteromers led to the same ketone, which was gem-dimethylated using MeI and sodium hexamethyldisilazide (NaHMDS). Chemoselective hydrogenation with Adam’s catalyst, and

O N

Boc 39

65% over 3 steps h) TFA; HO TsCl i) TBAF j) Li/NH 3 O k) tBuOK, PhCOPh Me

AcO Me

O

H

N TBSO

Boc

Me

4 steps

NMeBoc OH OTBS 37

NMe

N

TBSO 40

Me

36

f) ZnBr2, 1-dodecanethiol

O

O

O

Me

TBSO

O AcO

O

d) TMAD, 38, PBu 3 e) MePPh 3Br n-BuLi

HO

g) Pb(OAc) 4

Br CO2K

Me Me 80% over 3 steps

CHO

38

N

MeO OMe

OH c)

MOMO

Figure 2. Catalytic cycle of Rieske dioxygenase and features of the dihydroxylated product that contribute to its synthetic versatility.

N

41

O 16% Boc over 4 steps ent-hydromorphone (33)

Ent-Hydromorphone Due to their biological properties, morphinan alkaloids have been the subject of many total synthesis efforts. Hudlicky et al. recently developed an efficient chemoenzymatic route to ent-hydromorphone (33, Scheme 5).35 The sequence began with an enzymatic dihydroxylation

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Biochemistry

of 2-phenylethylbromide (34) using whole cell fermentation to give diol 35 which was selectively reduced and protected to afford acetonide 36. Subsequent displacement of the bromide with methylamine and protecting group manipulations gave the key fragment 37, which was coupled with phenol 38 under Mitsunobu conditions. Following Wittig olefination and MOM group deprotection, the phenol moiety was oxidized with lead tetraacetate to yield the Diels–Alder precursor 40, which underwent the cycloaddition exclusively as the exocyclic diene to give compound 41 in 50% yield. Hudlicky and coworkers hypothesized that the low yield of the reaction was a result of only one diastereomer participating in the [4+2] cycloaddition. Notably, only a very small amount of product arising from cycloaddition at the endocyclic diene was observed, even though this motif is highly reactive. This observation was rationalized by invoking steric factors, which prohibit the dienophile from coming in proximity to the endocyclic diene. Rearomatization with TFA, tosylation, TBS deprotection, and cyclization via a nitrogen center radical afforded the pentacyclic core of ent-hydromorphone. The final oxidation was achieved using modified procedures from Woodward and Rapoport.36 Future Directions The examples we have presented in the preceding sections highlight the transformative power of biocatalytic oxidation in simplifying challenging problems in complex molecule synthesis. From the perspective of synthesis design, the general strategy for developing a successful chemoenzymatic synthesis using enzymatic hydroxylation typically commences with the identification of a strategic C–O bond to disconnect in the target molecule. Next, potential enzyme candidates for the oxidative transformation are identified on the basis of known substrate promiscuity profile or potential structural resemblance between the native and proposed substrate. Once these are established, the remaining gaps in the proposed route leading to the oxidation substrate are filled out using conventional retrosynthetic analysis. Despite these successes, chemoenzymatic total synthesis remains a highly specialized subject, comprising only a small fraction of total activity in the field of natural product synthesis. Moreover, oxidative enzymes that have been employed for synthetic applications have mostly been confined to the P450 and arene dioxygenase families. This situation stands in stark contrast to the rapid pace of development in the area of enzyme discovery. Aided by advances in genomics, our capability of identifying new pathways for secondary metabolite biosynthesis has expanded dramatically, which inevitably leads to the discovery of new enzymes with novel reactivity profiles. Concurrently, new techniques in enzyme engineering have allowed us to rapidly optimize the catalytic activity as well as tailor the selectivity profile of any given enzyme. In order to push the field of chemoenzymatic synthesis forward, it is imperative that we capitalize on these technologies and begin exploring the synthetic applications of other families of oxidative enzymes. The next few examples represent untapped discoveries of novel and/or engineered oxygenases with potentially broad-ranging applications in complex molecule synthesis. To illustrate their enabling power, we have added potential scenarios in which these enzymes could greatly facilitate synthetic access to a range of bioactive natural products.

discovery of new P450BM3 variants for the hydroxylation of a wide range of terpene scaffolds with complementary regioselectivity.38 One of the key discoveries made during this campaign is the identification of several P450BM3 variants capable of selective C3 hydroxylation of sclareolide (42), which stands in stark contrast to the lack of C2:C3 regioselectivity obtained using available chemical methods.39 Sclareolide (42) has recently gained attention as a viable starting point for chiral pool synthesis due to the prevalence of the trimethyldecalin motif in many higher order terpenes.40 This approach has resulted in the successful syntheses of various terpenoids such as pelorol40a and hongoquercin,40b and more recently the combination of this approach with selective C2 functionalization of sclareolide has resulted in the syntheses of 2-oxoyahazunone41a and lissoclimide.41b We anticipate that the ability to selectively perform C3 oxidation on sclareolide will open up new avenues for the chemoenzymatic synthesis of terpene natural products. For example, 3-hydroxysclareolide (43) can be subjected to further functionalization with Hartwig’s directed C–H silylation42 and subsequent oxidation to afford the corresponding C15 oxidized product (Scheme 6). Next, the butyrolactone moiety can be activated for subsequent cross-coupling with an appropriately-functionalized indole to afford requisite connectivity of indosespene (45). Finally, further oxidation and electrocyclization will generate xiamycin A (46).43 As an alternative to Ir-catalyzed C–H silylation, biocatalytic oxidation with XiaM,44 a P450 from the xiamycin biosynthesis pathway, could also be utilized for the formation of the carboxylic acid moiety of the target natural product. Scheme 6. A. Proposed application of enzymatic hydroxylation of sclareolide with an engineered P450 en route to the chemoenzymatic synthesis of xiamycin A. B. Select examples of natural products containing oxidized trimethyldecalin motif. A

O Me

O

H

Me

Me Me sclareolide (42)

O

O

P450 BM3 "II-H8"

Me

P450 oxidation HO

O

H Me

C–H Silylation

Me H

Me O R 2Si

Me 43

O Me

Me 15

44

Me H HO Me

Me

NH

NH

oxidation

XiaM

Me Me H HO HO 2C

Me

H HO HO 2C

Me

H HO HO 2C

Me xiamycin A (46)

B

-O

Me 45

3SO

OMe

Me O Me

O

Me OSO 3-

Me Me

Me

H

H

Me

O H

Me

H

H adociasulfate-13

HO

HO Me

O Me

Me

arisugacin F

P450s We have previously highlighted the use of a P450BM3 mutant in a latestage oxidation approach towards nigelladine A (16). P450BM3 has garnered tremendous interest for biotechnological uses due to its selfsufficient nature and high evolvability.37 Pioneering work by Arnold has provided the blueprint for the engineering of this enzyme for a multitude of applications, ranging from lead compound modification, monosaccharide manipulation, to chemomimetic biocatalysis. Recently, the Fasan laboratory has developed a rapid fingerprinting method for the

Flavin-Dependent Monooxygenases Flavin-dependent monooxygenases (FMOs) are defined by their ability to catalyze substrate specific oxidation utilizing molecular oxygen, NAD(P)H and a flavin cofactor.45 At least 130 FMOs are known, subdivided into six divisions defined by sequence, structure, and function. FMOs can perform a broad array of chemical transformations, including hydroxylation, Baeyer-Villiger oxidation, sulfoxidation, epoxida

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Scheme 7. Proposed use of TcmG-catalyzed arene oxidation in the chemoenzymatic syntheses of tetracenomycin C and viridicatumtoxin B. O

OH

Me

see ref. 51

Cl

OH

O

49

OH

Me

4a

OMe

4

O tetracenomycin A2 (48) TcmG FMO oxidation OH

O

MeO 2C

Me O O

Me

OH

O

MeO 2C

Iron- and α-Ketoglutarate-Dependent Dioxygenases

O OH 12a

4a

O Me MeO

OH

O

MeO

O

OH OH

OMe

MeO

OMe

4

OH

elloramycin A

O

OH OH

tetracenomycin C (47)

OMe O

OH

OH

OH

O

OH

Cl

CONHR MeO O

OH

OH

Br

49 FMO oxidation

Br Me

53

OH

c) Pd cat., [Zn], 53 d) BF 3•Et 2O

Me

TBSO

O 51 a) TcmG b) reduction

OH

O

O OH

Me

e) Deprotection

MeO Br

O

CONHR OH

OH

52 OH

MeO Me

OH

O

O

O OH

OH

CONH2 OH

Me viridicatumtoxin B (50) Me

stallation should yield 51; which would prove an interesting molecule to probe TcmG’s substrate scope. Allylic bromide 53 has recently been used in the total synthesis of viridicatumtoxin B, and should prove amenable to complete the synthesis through Negishi cross-coupling and acid-catalyzed spirocyclization.53

12a

MeO 2C MeO

O

OH

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The iron- and α-ketoglutarate-dependent dioxygenases (Fe/αKGs) are defined by the presence of Fe-binding His1-X-Asp/Glu-Xn-His2 motif and the use of α-ketoglutarate as a co-substrate in the reaction.43 During catalysis, activation of molecular oxygen results in the formation of a reactive Fe(IV)-oxo intermediate (Figure 3), with concomitant expulsion of CO2 from α-ketoglutarate to form Fe-bound succinate. Finally, substrate oxidation and release of succinate regenerate the resting state of the enzyme. The first Fe/αKG activity was identified in the hydroxylation of proline residues in collagen.55 Since then, many other members of the family have been identified and characterized, with an astonishing breadth of substrate diversity, including proteins, polynucleotides, amino acids, terpenoids, and alkaloids.56 Unlike the P450s and the Rieske oxygenases, Fe/αKGs do not require any dedicated reductase partners to perform their function. Despite early success in the use of enzymes from this family in the production of β-lactam analogs,57 other members have received only very little attention58 for synthetic applications. Given their self-sufficient nature, we anticipate that this enzyme family could provide a wide range of untapped opportunities for biocatalyst development for chemical synthesis.

R MeO

N

MeO

N

N

O

OH 2

NH

Glu/Asp His

O flavin cofactor

tion, halogenation, C–C bond formation, and structural rearrangements.46 While position selective arene hydroxylation is particularly valuable for the synthesis of fine chemicals and natural products, this feat is difficult utilizing traditional chemical methods.47 On the other hand, FMO enzymes have a unique ability to perform position selective arene hydroxylation with ease on a variety of substrates. Hydroxylated arenes are present in a diverse array of natural product families such as meroterpenoid, lignin, and polyketide.48 Combining chemical and enzymatic methods has potential to enable rapid access to these structurally interesting and biologically relevant natural product families. One potential application could be found in the chemoenzymatic total synthesis of the structurally unique polyketide antibiotic tetracenomycin C (47, Scheme 7).49 In 1994, biosynthetic studies on tetracenomycin C revealed that the FMO TcmG is responsible for introducing tetracenomycin C’s triply hydroxylated cyclohexanone motif, utilizing tetracenomycin A2 (48) as its substrate.50a Subsequent mechanistic studies confirmed the origin of each hydroxyl group introduced by TcmG: C4/C12a from two molecules of O2 and C4a from one molecule of H2O.50b A chemoenzymatic synthesis can be envisioned following the work of Cameron and co-workers, who previously published a total synthesis of tetracenomycin A2 in only 4 steps starting from 49 (Scheme 7).51 Following this synthetic route to tetracenomycin A2, TcmG-catalyzed oxidation could subsequently allow expedited access to tetracenomycin C. A similar approach may prove viable in the synthesis of the fungal antibiotic viridicatumtoxin B (50).52 Viridicatumtoxin B is a fungal toxin isolated from Penicillium viridicatum, consisting of a spirocyclic monoterpene fused to a naphthacenedione tetracycle. Consecutive Diels-Alder transformations and bromine in-

αKG, H 2O

Fe II

O O

O

R1 H

H 2O

R2

His His-X-Asp/Glu-X n-His motif R 1 = substrate CO2H

R2 =

R1

R1 O

OH Glu/Asp His

H R2

Glu/Asp His

Fe II O His

Fe II

O O

O R2

His

R1

O2

R1 H OH

Glu/Asp His

O

Fe III O His

R2

O Glu/Asp His

CO2

O R2

Fe IV O His

Figure 3. Catalytic cycle of Fe/αKG hydroxylation utilizing αketoglutarate as a co-substrate. One intriguing application of Fe/αKG hydroxylation could be found in the use of desacetoxyvindoline 4-hydroxylase (D4H) in the synthesis and diversification of Vinca alkaloids. The Vinca alkaloids are a diverse family of terpene indole alkaloids with fascinating molecular architectures and biological activities.59 Several members of the family, including vinblastine and vincristine, have found clinical applications as antineoplastic drugs. Elucidation of the biogenesis of these alkaloids in plants has revealed the presence of D4H,60 an Fe/αKG responsible for

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Biochemistry

the oxidation of the C4 carbon en route to the production of vindoline (54). Subsequent work by De Luca and co-workers has resulted in heterologous expression and in vitro characterization of this enzyme.61 Based on this precedent, a chemoenzymatic synthesis of vindoline can be envisioned (Scheme 8), which would commence with an efficient skeletal construction employing Boger’s cycloaddition cascade strategy62 to afford 56. Short chemical manipulations, followed by a latestage enzymatic oxidation and acetylation can then be employed to obtain vindoline (54). In addition, engineering D4H for complementary hydroxylation regioselectivity could lead to rapid scaffold diversification, which would pave the way for the development of novel vinblastine analogs. Scheme 8. Proposed use of D4H-catalyzed hydroxylation in the chemoenzymatic total synthesis of vindoline. X

X N N

O MeO

N MeO

X = O or S

N

N Me

see ref. 62 Et

Me

CO2Me 55

MeO Me

4

N

H Me OH MeO 2C

4

H 56

N

OAc

a) D4H, αKG, Fe 2+, O2 MeO b) Ac2O Fe/αKG hydroxylation

vindoline (54)

Me

O N

CO2Me

N Me 4

N

H Me OH MeO 2C 57

Me

HO N N N H MeO 2C MeO

NH R1

vinblastine: R1 = Me, R 2 = OMe, R 3 = Ac Me vincristine: R1 = CHO, R 2 = OMe, R 3 = Ac vindesine: R1 = Me, R 2 = NH 2, R 3 = H OR 3 OH COR2

Conclusion The post-genomic era has seen a dramatic rise in the discovery of novel enzymes from secondary metabolite biosynthesis pathways. However, the rate of discovery of new enzymes is incommensurate with their adaption as biocatalysts, especially for synthesis of complex targets. To date, the field of chemoenzymatic synthesis is still perceived as a “niche” area of research, with relatively high barriers to entry (vide infra). In this regard, some parallels can be drawn to the field of chemical C–H functionalization.63 Early discoveries in C–H activation by Fujiwara,64a Bergman,64b Murai,64c and others were initially deemed as impractical, impeding substantial progress for more than a decade. It was not until the initial successes of implementing C–H functionalization in complex molecule synthesis that subsequent developments began to emerge, leading to the design of new catalysts and innovative directing groups to address selectivity issues of the reaction. Owing to these advances, C–H functionalization has revolutionized the way chemists approach synthetic planning, with numerous reviews and tutorials published to guide strategic applications of this paradigm.63,65 Similarly, oxidative enzymes, by virtue of their exquisite selectivity profile, have the potential to transform contemporary logic in chemical synthesis. However, such potential has remained largely unrealized. Natural product synthesis is often regarded as the ultimate testing ground for synthetic methods and the tepid reception for biocatalytic oxidation is readily apparent here as case studies of chemoenzymatic

total synthesis featuring enzymatic oxidation, while highly fascinating, remain few and far between. In order to bridge this gap, we believe several challenges will need to be addressed. Enzymatic catalysis is still viewed as impractical as transformations are often conducted only on analytical (milligram) scale employing chromatographic assays without product isolation. Thus, performing preparative reactions on gram scale during the discovery process can greatly aid in dispelling this notion. The issue of practicality is directly tied to the protein expression level and the catalytic performance of a given enzyme. Due to its high molecular weight, a given enzyme needs to be able to achieve high total turnover numbers for the biotransformation to be practical. Similarly, the high substrate specificity of an enzyme can also be its downfall as this property can result in a narrow substrate scope. For both of these issues, methods of directed evolution and enzyme engineering could provide viable options to boost the desired enzymatic activity and expand the substrate scope. 66 Of course, these methods come with their own set of challenges and the readers are cautioned against viewing enzyme engineering as a panacea for the aforementioned limitations. Some enzymes are inherently more promiscuous and/or evolvable than others58 and at present, we have little understanding of the various factors that contribute to these properties. Compounding the issue at hand is the fact that oxidation of complex natural product scaffolds or synthetic intermediates thereof do not always allow for high throughput (fluorescent/colorimetric) readout and often require the use of “specialized” oxygenases from secondary metabolite pathways with minimal sequence-function information. As such, formulating an efficient directed evolution platform can be problematic. Though these challenges are significant, recent successes in incorporating computational tools67 and new analytical techniques68 are encouraging, and could lay the groundwork for further integration of new technologies to address some of the bottlenecks of directed evolution. Finally, the lack of ready access to enzyme catalysts represents another obstacle to a wider adoption. The process of obtaining an enzyme catalyst typically involves the construction of an appropriate expression vector, followed by in-house enzyme overexpression and purification with fast protein liquid chromatography (FPLC) systems. Once obtained, such an expression vector and the corresponding enzyme are kept in house within the producer laboratory. Thus, most oxidative enzymes are not available from commercial vendors and laboratories with no prior experience in bacterial cell culture and protein purification are less inclined to incorporate these catalysts in their work. While we are still years away from a practical cell-free transcriptiontranslation kit69 for biocatalysis, an alternative solution could be found in the development of a reliable lyophilization and/or immobilization protocols70 and subsequent industrial partnership that would allow the dissemination of new biocatalysts to a wider audience. We believe that commercial availability will contribute greatly towards demystifying enzyme as another catalyst that is not too dissimilar from a conventional organometallic or organocatalyst. Alternatively, collaborative efforts can be pursued to highlight the synergy of enzymatic oxidation and contemporary chemical methods, and recent success20 from Arnold and Stoltz highlights the potential of this approach. While the challenges outlined here are substantial, advances in biotechnology and biochemical techniques have provided us with the necessary foundation to bring the synthetic utility of oxidative enzymes to the fore. With continued interdisciplinary collaborations between synthetic chemists, biologists, and engineers, we believe that the field of biocatalytic C–H oxidation can flourish to provide practical tools for the synthesis of complex molecular targets.

AUTHOR INFORMATION Corresponding Author

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[email protected]*

24.

Author Contributions

25. 26.

‡These authors contributed equally.

ACKNOWLEDGMENT

27.

We gratefully acknowledge TSRI for financial support. We thank W. R. Gutekunst and T. R. Newhouse for helpful discussions in manuscript preparation.

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R H

feedstock chemicals

R OH

enzymatic C–H hydroxylation

OH

HO

OH

bioactive natural products

organic reactions

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