Enzymatic Cascade Reactions in Biosynthesis

Abstract

Enzyme-mediated cascade reactions are widespread in biosynthesis. To facilitate comparison with the mechanistic categorizations of cascade reactions by synthetic chemists and delineate the common underlying chemistry, we discuss four types of enzymatic cascade reactions: those involving nucleophilic, electrophilic, pericyclic, and radical reactions. Two subtypes of enzymes that generate radical cascades exist at opposite ends of the oxygen abundance spectrum. Iron-based enzymes use O₂ to generate high valent iron-oxo species to homolyze unactivated C-H bonds in substrates to initiate skeletal rearrangements. At anaerobic end, enzymes reversibly cleave S-adenosylmethionine (SAM) to generate the 5'-deoxyadenosyl radical as a powerful oxidant to initiate C-H bond homolysis in bound substrates. The latter enzymes are termed radical SAM enzymes. We categorize the former as "thwarted oxygenases".

Keywords: electrophilic cascades; natural products; nucleophilic cascades; pericyclic cascades; radical cascades.

Scheme 1

Examples of natural product chemical syntheses exemplifying the five mechanistic categories of cascade reactions. A) A nucleophilic cyclization cascade in the total synthesis of tetronasin by Ley and colleagues.^[7] B) An electrophilic cascade involving an epoxy-olefin cyclization in the total synthesis of hemibrevetoxin B by Holton and colleagues.^[8] C) A radical cyclization cascade in the total synthesis of morphine by Parker and Fokas.^[9] D) A pericyclic cascades involving Diels-Alder and [3+2] cycloadditions in the total synthesis of vindorosine by Boger and colleagues.^[10] E) Transition-metal-catalyzed cascades involving multiple ring-opening/ring-closing olefin-metatheses in the total synthesis of cyanthiwigin U by Pfeiffer and Phillips.^[11] AIBN =2,2’-azobisisobutyronitrile, KHMDS =potassium bis(trimethylsilyl)amide, MOM =methoxymethyl, N-PSP =N-(phenylseleno)phthalimide, TIPB =triisopropylbenzene, Ts =p-tolylsulphonyl.

Scheme 2

Two polyketides generated by enzymatic cascades. A) The 14-member deoxyerythronolide B (DEB) lactone is released from the three-subunit DEB synthase assembly line after incorporation of seven methylmalonyl units. B) Oxytetracycline biosynthesis involves a chain elongation cascade to a nineteen carbon nonaketonyl thioester that is converted into the tetracyclic pretetramide product.

Scheme 3

Nonribosomal peptide synthetase assembly lines carry out chain-elongation cascades to the heptapeptide vancomycin (A) and the hybrid NRPS/PKS product rapamycin (B), in which amine nucleophiles are utilized for the insertion of amino acids.

Scheme 4

The fumiquinazoline F NRPS assembly line carries out a cascade employing the amino group of the anthranilyl-1 residue for cyclizing release, followed by transannular capture of the transient 6,0-macrocycle to yield the tricyclic quinazolinone framework.

Scheme 5

Oxa-1,4-cojugate additions create pyran rings on the PKS assembly lines during pederin (A), ambruticin S (B), and salinomycin (C) polyketide assembly.

Scheme 6

The antibiotic lugdunin arises from an NRPS assembly line cascade. The release step involves reduction of tethered peptidyl thioester by hydride transfer from NADH catalyzed by the LugC terminal reductase domain. The released aldehyde can circularize as the cyclic imine, which is further driven to accumulate as the cyclic thiazolidine from addition of the cysteine thiolate to the imine.

Scheme 7

The enzyme SAH lyase starts a cascade leading to thioether cleavage by initial hydride-mediated oxidation of the ribose-3-OH to the ketone. This lowers energy for C₄ carbanion as the internal nucleophile required for C₅–S cleavage.

Scheme 8

Reductive strategies to carbocycle formation in plant iridoid scaffolds (A) and bacterial polycyclic tetramate macrolactams (B).

Scheme 9

The redox step catalyzed by the FAD-containing solanapyrone synthase brings the aldehyde into conjugation with the exocyclic olefin to increase its reactivity as a dienophile for the ensuing Diels-Alder cyclization.

Scheme 10

The FAD-enzyme that catalyzes a Baeyer-Villiger oxygenation on the d-ring of tetracyclic premithramycin converts the cylohexenone D-ring into the ring-expanded lactone. This is now labile to water-mediated hydrolytic opening. The released enediolate isomerizes to the tricyclic ketone mithramycin product.

Scheme 11

The EncM flavoenzyme doubly oxygenates a 1,3-diketo moiety of the enzyme-bound poly-β-keto intermediate to a 1,2,3-triketo nascent product via the newly discovered flavin-N5-oxide cofactor. This product intermediate is subject to a Favorskii-type nucleophilic rearrangement cascade with formation of a transient hydroxycyclopropanone.

Scheme 12

A sequiterpene cation rearrangement cascade converts the acyclic C₁₅ farnesyl-diphosphate into the tricyclic-5,5,5-framework of pentalenene during the biosynthesis of pentalenolactone.

Scheme 13

A diterpene rearrangement cascade converts acyclic C₂₀ geranylgeranyl-diphosphate into tricyclic taxadiene.

Scheme 14

Triterpene cyclization cascades. A) Squalene-hopene cyclase initiates the electrocyclic cascade by protonation of the 2,3-terminal double bond of the acyclic C₃₀ hexaene squalene. B) Cyclization of squalene to lanosterol via a cascade of cation, hydride, and methyl group migrations after oxidosqualene formation. C) Enzymatic cyclization cascade from 2,3-epoxysqualene to the pentacyclic β-amyrin product is the predominant mode in plant metabolism.

Scheme 15

Enzymatic C-methylation at an olefin initiates an electrophilic cascade in teleocidin B biosynthesis.

Scheme 16

Enzymatic double chlorination of a naphthol ring by chloronium ion equivalents sets a carbocation cascade in motion in merochlorin A and B formation. Merochlorin C formation rather involves a Cl⁺-mediated α-hydroxyketone rearrangement and a third chlorination event.

Scheme 17

Baruol synthase leaks a set of minor products reflecting capture of intermediates at different points in the cationic cascade process. Bold arrows show the primary route to baruol with representative pathway byproducts shown to other cyclic triterpenes with relative percentage product distribution.

Scheme 18

Disappearing-epoxide cascade reactions in the enzymatic conversion of 3-prenylindole substrates into paspaline (A) and xiamycin (B).

Scheme 19

Tandem action of the epoxidase Lsd18 and “epoxide hydrolase” Lsd19 convert a bis-olefin by way of bis epoxide into the tetrahydrofuran and tetrahydropyran rings of the ionophore lasalocid A.

Scheme 20

It is proposed that 11 double bonds are converted into 13 epoxides as precursors to the fused cyclic ethers in the potent marine ciguatoxin.

Scheme 21

Three disappearing epoxide intermediates in assembly of the bicycloctane scaffold of the ATP synthase inhibitor aurovertin.

Scheme 22

Tandem action of PyrE3 and PyrE4 as catalysts of two types of Diels-Alder cyclization in the biosynthetic pathway to pyrroindomycin

Scheme 23

SpnF and SpnL build the fused 5,6,5-tricyclic core of the insecticidal agent spinosyn through Diels-Alder and Rauhut-Currier reactions.

Scheme 24

Biosynthesis of the trithiazolylpyridine core of thiocillin-type antibiotics. Proposed aza-Diels-Alder cyclization in the reaction catalyzed by ThiM during thiocillin assembly. Creation of the pyridine ring at the core of the trithiazolylpyridine array also closes the 26-membered macrocyclic ring in thiocillin.

Scheme 25

The enzyme LepI carries out both a conventional [4+2] cyclization and an oxa-[4+2] cyclization competitively. The latter reaction yields the main product leporin C. The spiro product from the conventional [4+2] pathway is then subjected to an enzymatic [3,3] retro-Claisen reaction to rescue the stranded material and convert it into leporin C.

Scheme 26

Chorismate mutase catalyzes the only known [3,3]-rear-rangement in primary metabolism of microbes and plants.

Scheme 27

Proposed Cope rearrangement in the biosynthesis of the brown algal feeding deterrent ectocarpene from a transient cyclo-propane pheromone, which in turn arises from peroxidative fragmentation of a polyunsaturated fatty acyl peroxide.

Scheme 28

Hapalindole and fischerindole biosynthesis arise via a [3,3]-Cope rearrangement followed by an aza Prins reaction.

Scheme 29

Two enzymatic strategies to homolyze unactivated C–H bonds in substrates and create transient carbon-centered radicals involving oxygenative and nonoxygenative paths. A) Two variants of iron-based oxygenases, namely heme-containing cytochrome P450s and mononuclear nonheme iron enzymes that require co-substrate a-ketoglutarate, generate high-valent Fe^V=O and Fe^IV=O oxidants, respectively, for co-substrate C H bond homolysis. B) The strategy for homolytic cleavage of S-adenosylmethionine (SAM) in radical SAM enzymes by one-electron transfer from a 4Fe/4S cluster to generate the 5’-deoxyadenosyl radical (dA^•) as initiator of substrate C–H bond homolysis.

Scheme 30

Isopenicillin N synthase (IPNS) is a “thwarted oxygenase” that converts the acyclic tripeptide ACV into the fused 4,5-ring system of the penicillin family of β-lactam antibiotics through a cascade of radicals.

Scheme 31

The vancomycin heptapeptide is tailored on the NRPS assembly line by three dedicated P450 enzymes. Each acts as a thwarted oxygenase, generating carbon-based radicals in the electron-rich aromatic side chains of the heptapeptide and ultimately producing two aryl ether crosslinks (C-O-D and D-O-E aryl ether bonds) and one direct C–C link (A-B biaryl linkage).

Scheme 32

The P450-containing GsfF enzyme catalyzes a radical-based reaction pathway to create the spirocyclic ring system of the fungal metabolite griseofulvin in a “thwarted oxygenase” mode.

Scheme 33

Additional enzyme-mediated radical redirection reactions in the biosynthesis of the alkaloid salutaridine (A), the indolecarbazoles staurosporine and rebeccamycin (B), and the diketopiperazine fumitremorgin C (C).

Scheme 34

The biosynthesis of communesin B involves the thwarted P450 oxygenase CnsC, which mediates coupling between the tryptamine and aurantioclavine radical partners.

Scheme 35

P450 CYP88A-mediated conversion of ent-kaurenoic acid into giberellic acid-12-aldehyde via a reaction sequence onvloving a radical-based ring contraction and aldehyde carbon -C6H^•(OH) extrusion concomitant with oxygen transfer.

Scheme 36

Enzyme mediated O₂-dependent radical cascades in the late stages of the biosynthesis of anditomin meroterpenoids. AndA creates a bicyclooctane ring embedded in the meroterpenoid scaffold, while AndF introduces the final nonoxygenative radical rearrangements in the 12-enzyme pathway to anditomin.

Scheme 37

Enzymatic assembly of the octacyclic framework of the insect ion channel blocker okaramine E in a short, efficient pathway involving two thwarted oxygenases, the P450 OkaD and the nonheme iron enzyme OkaE, that catalyze radical cascade reactions to generate the eight-member azocine ring and the four-member azetidine ring, respectively.

Scheme 38

Phenylpropanoid metabolite O₂-dependent diverted radical cascades. A) Oxidative dimerization of coniferyl alcohol to different lignan frameworks. B) Enzymatic flux of flavonoids to isoflavonoids by 1,2-aryl migration in a radical intermediate.

Scheme 39

Two radical SAM enzymes in the anaerobic aminofutalosine pathway to menaquinone (vitamin K).

Scheme 40

In hydrogenase activation, a radical SAM enzyme supplies CO and CN as ligands for the active-site iron through radical fragmentation of substrate L-tyrosine.

Scheme 41

Synthesis and biosynthesis of the 7,7-para-cyclophane cylindrocyclophane F by different cascade strategies. A) Synthesis by a double metathesis cascade. B) Biosynthesis by an apparent Friedel Crafts type bis alkylation cascade to create the para-cyclophane macrocycle.