Enzymatic Cascade RX

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International Edition: DOI: 10.1002/anie.201807844

Biosynthesis German Edition: DOI: 10.1002/ange.201807844
Enzymatic Cascade Reactions in Biosynthesis

Christopher T. Walsh and Bradley S. Moore*
Keywords:
electrophilic cascades ·
natural products ·
nucleophilic cascades ·
pericyclic cascades ·
radical cascades
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Enzyme-mediated cascade reactions are widespread in biosynthesis. From the Contents

To facilitate comparison with the mechanistic categorizations of
1. Introduction to Cascade
cascade reactions by synthetic chemists and delineate the common
Reactions in Total Synthesis 3
underlying chemistry, we discuss four types of enzymatic cascade
reactions: those involving nucleophilic, electrophilic, pericyclic, and 2. Enzymatic Cascades 3
radical reactions. Two subtypes of enzymes that generate radical
cascades exist at opposite ends of the oxygen abundance spectrum. 3. Enzymatic Nucleophilic
Cascades on Assembly Lines 5
Iron-based enzymes use O2 to generate high valent iron-oxo species to
homolyze unactivated C H bonds in substrates to initiate skeletal 4. Enzymatic Electrophilic
rearrangements. At anaerobic end, enzymes reversibly cleave S- Cascades 11
adenosylmethionine (SAM) to generate the 5’-deoxyadenosyl radical
5. Pericyclases 17
as a powerful oxidant to initiate C H bond homolysis in bound
substrates. The latter enzymes are termed radical SAM enzymes. We 6. Radical-Based Cascades 22
categorize the former as “thwarted oxygenases”.
7. Cylindrocyclophane:
Nonenzymatic Metathesis
Versus Apparent Friedel–Crafts
Enzymatic Bis-alkylation 30
1. Introduction to Cascade Reactions in Total 8. Summary and Outlook 31

Synthesis
Cascade reactions have attracted special attention both

historically and among current organic chemists for both have completed total syntheses. Some readers may wish to go
intellectual and practical challenges (e.g. atom economy[1] and back and compare natures actual cascade strategies with
synthetic ideality[2]) in the generation of molecular scaffold those of synthetic chemists, who proceeded, with or without
complexity.[3] Cascade reactions, both planned and somewhat a preconceived notion of biomimicry, often before the
serendipitous, have advanced mechanistic understanding and relevant enzymes were even identified and characterized.
molecular reactivity, for example, Robinsons tropinone This rubric seems a useful starting point as well for the
synthesis in 1917,[4] the polyolefin cyclization of Johnston enzyme-mediated cascades, since both enzymatic and non-
and colleagues in the synthesis of progesterone in 1971,[5] and enzymatic synthetic transformations follow the same rules of
many dozens over the past five decades.[6] organic chemistry, although there are no known equivalents
Nicolaou and colleagues[3a] authored an extensive review of olefin metathesis reactions (yet) in nature. Thus, we present
on Cascade Reactions in Total Synthesis in 2006 that collected four categories of nucleophilic, electrophilic, pericyclic, and
many examples of strategies for the assembly of several radical-based cascades in enzymatic catalysis.
natural product scaffolds in which cascade reactions of
different types created dramatic increases in framework
complexity. They often represent the apotheosis of both 2. Enzymatic Cascades
skill and elegance as organic chemists use synthetic campaigns
as proving grounds for understanding and predicting reac- For more than a century, it has been clear that proteins
tivity rules. with catalytic activity, enzymes, carry out essentially all of the
Cascade reactions transform reactants into intermediates/
products in multiple distinct steps. Thus, the definition of
[*] Prof. Dr. B. S. Moore
where a cascade stops may be the practical stage of a one-pot Center for Marine Biotechnology and Biomedicine
reaction. This is a different endpoint from the enzymatic cases Scripps Institution of Oceanography
discussed in this Review, where release of a product from the University of California, San Diego, La Jolla, CA 92093 (USA)
active-site microenvironment is the reaction endpoint. and
Nicolaou and colleagues[3a] also noted that the nature of Skaggs School of Pharmacy and Pharmaceutical Sciences
multistep cascade transformations can complicate classifica- University of California, San Diego, La Jolla, CA 92093 (USA)
E-mail: bsmoore@ucsd.edu
tion but they divided their analyses into five mechanistic
Prof. Dr. C. T. Walsh
categories: nucleophilic, electrophilic, radical-based, pericy-
Stanford University Chemistry, Engineering, and Medicine for
clic, and transition-metal-based mechanisms for the frame- Human Health (CheM-H), Stanford University
work rearrangements (Scheme 1). Stanford, CA 94305 (USA)
Readers are referred to that review and related articles.[6] The ORCID identification number(s) for the author(s) of this article
Many of the end products of enzymatic cascades noted here can be found under:
have attracted the attention of natural product chemists who https://doi.org/10.1002/anie.201807844.
Angew. Chem. Int. Ed. 2019, 58, 2 – 36 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org &&&&
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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.
controlled chemical transformations in living organisms. In an remnants of which exist today, for example, in the ribonu-
RNA-world scenario, they were preceded by catalytic RNAs, cleoprotein ribosomes that carry out protein biosynthesis.[12]
Christopher Walsh is a consulting professor Bradley Moore is Professor of Marine Chem-

to the Stanford University department of ical Biology at the Scripps Institution of
chemistry and an advisor to the Stanford Oceanography and Chair of Pharmaceutical
CheM-H Institute. He was Chemistry Chemistry at the Skaggs School of Phar-
Department Head at MIT and the Hamilton macy and Pharmaceutical Sciences at UC
Kuhn Professor of Biological Chemistry and San Diego. He received his B.S. in chemistry
Molecular Pharmacology at Harvard Medi- from the University of Hawaii, his PhD from
cal School from 1987—2013. His research the University of Washington, and was
has explored the chemical mechanisms used a postdoc at the University of Zurich. His
in nature to build complexity into natural research focuses on understanding how
product scaffolds. microbes and marine organisms construct
complex bioactive molecules with an eye
toward biomedicine and biotechnology.
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There are some cascade reactions in primary metabolism, the nonribosomal peptide/polyketide natural product frame-
interconversion of small-molecule metabolites into product- works. A large subset of polyketide synthases (PKSs) and
(s) carried out by all cells. For example, the tandem action of essentially all of the nonribosomal peptide synthetases
thiolase and hydroxymethylglutaryl-CoA synthase converts (NRPSs) hold onto the growing intermediate chains as
three acetyl-CoA molecules to the C6 half thioester HMG- covalent thioesters and thus qualify as cascade catalysts.[13]
CoA.[13] However, the major enzymatic complexity-generat- Carbanion equivalents as nucleophiles. The celebrated
ing events occur in conditional pathways, known collectively deoxyerythronolide B (DEB) synthase, for example, takes
as secondary metabolism. seven molecules of 2S-methylmalonyl-CoA and elongates
This is the realm of natural product biosynthesis, that is, them to the 14-carbon linear thioester and then carries out
biosynthesis of the isoprenoids, alkaloids, phenylpropanoids, regiospecific cyclization to release the 14-member DEB
polyketides, and nonribosomal peptides that account for most macrocyclic lactone as only one of 1024 possible diastereo-
of the approximately 500,000 known natural products.[13] mers[17] (Scheme 2 A). Analogously, in the biosynthesis of
These also include the bioactive molecular frameworks that tetracycline, the enzyme trio of OxyABC builds a tethered 19-
have been the targets of hundreds of natural product synthetic carbon nonaketidyl thioester chain (Scheme 2 B). Tailoring
studies by chemists over the past 80 years. enzymes then carry out transannular aldol and Claisen
Cascade reaction planning, implementation, and analysis condensation reactions to release preteramid as the first
have been core activities of synthetic chemists in the context soluble intermediate.[18]
of biomimetic or “bioinspired” routes.[3a, 6] Many of these To build these nucleophilic chain-elongating cascade
synthetic campaigns were undertaken before the enzymes of reactions, polyketide synthases need carbon nucleophiles to
a particular pathway were identified, purified, and examined generate the C C bonds. These are typically the CoA
for mechanism and specificity. Nonetheless, they often had thioester enolates of malonyl and methylmalonyl acids (all
predictive power, given that chemical mechanisms play out in seven monomers incorporated in DEB above). The thioester
biology. Indeed, a recent review by Liu and co-workers has grouping activates C1 as the electrophile and adjacent C2 as
catalogued examples of dozens of organic named reactions in the nucleophile for iterative thioclaisen condensations (see
enzymatic catalysis,[14] many of which are key steps in both Ref. [13] for a review).
nonenzymatic and enzymatic synthetic cascades. Amines as nucleophiles. In contrast to polyketide cas-
In this Review, we delve into enzymatic examples of cades, where C C bond formations are the chain elongation
cascade reactions to illustrate how barriers for multistep steps, NRPSs make amide bonds and use the amino nitrogen
transformations are lowered in specific enzyme active sites. atoms of tethered amino acid monomers as the chain-
These are part and parcel of the ability of enzymes involved in elongating nucleophiles.[19] In the active sites of the conden-
natural product biosynthesis to build complexity from simple sation/chain elongation catalytic domains of NRPS assembly
primary metabolites. The coverage is not meant to be lines, the predominant -NH3+ ionization states must be
encyclopedic but rather to illustrate how some of the organic converted into nucleophilic NH2 groups (Scheme 3 A). Con-
named reactions are put to use in cascades within a given comitantly, in hybrid NRPS/PKS assembly lines, amines and
enzyme active site, natures equivalent of the one-pot thioester enolates are the requisite nucleophiles, for example,
synthetic reaction. We will mention a few tandem reactions, in rapamycin (Scheme 3 B) or FK506 assembly.[20]
involving the consecutive action of two enzymes to promul- An example of an unleashed cascade reaction occurs in
gate the cascades. We exclude multienzyme participations in the action of the fungal trimodular NRPS that makes
full biosynthetic pathway reconstitutions, although we have fumiquinazoline F, in which anthranilate, tryptophan, and
noted elsewhere the remarkable efficiency of enzymes to alanine building blocks are combined into a fused tricyclic
function in short pathways that build remarkable scaffold quinazoline core.[21] The assembly line enzyme builds a teth-
complexity.[13, 15] ered linear anthranilyl-Trp-Ala-thioester, which undergoes
While one could divide the presentation according to intramolecular capture by the anthranilyl amine, releasing
specific natural product categories, we think it more useful to a presumptive 6,10-macrocycle. This is never detected.
categorize enzymatic cascade reactions by mechanism. To Instead, one observes only the transannular, cyclodehydrated
that end we use the Nicolaou[3a] approach of nucleophilic, quinazoline product (Scheme 4).
electrophilic, radical-based, and pericyclic cascades. There is We note in the section on radical-driven cascades below
no enzymatic analogue of nonenzymatic olefin metathesis that the tethered heptapeptidyl chain in the vancomycin
cascades and no indication that palladium or platinum are synthetase assembly line is acted on successively by three
involved in biology. Molybdenum is used in a small number of “thwarted oxygenases” that make side-chain radicals that
enzymes[16] but not detectably for olefin metatheses. form cross links rather than undergo hydroxylations, as
emblematic of tailoring reactions that occur on NRPS
assembly lines. There are many examples of tailoring reaction
3. Enzymatic Nucleophilic Cascades on Assembly on PKS assembly lines as well, including Michael additions by
Lines side chain -OH groups on conjugated enoyl thioesters to form
cyclic ethers (Scheme 5).[22]
Cascade reactions initiated or carried out by nucleophiles
are common in the enzymatic assembly lines that build many
thousands of polyketide, nonribosomal peptide, and hybrid
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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.
3.1. Nucleophilic Enzymatic Cascades Enabled by Redox Steps adduct and then reductively releases it through hydride
transfer from the co-substrate NADH to yield the nascent
There are a number of cases where enzymes that carry out linear peptide aldehyde (Scheme 6). This can be cyclized
an oxidative or reductive change on a substrate/nascent through attack of the N-terminal Cys1 amino group on the
product uncover reactions that can be categorized as cascade Val7 aldehyde to give the cyclic imine. The equilibrium in
reactions. One simple case, extending the above discussion of favor of cyclization is further driven by addition of the Cys1
NRPS assembly lines, is the recent discovery of the pathway side-chain -SH onto the imine to yield the cyclic thiazolidine.
to the cyclic peptide lugdunin, produced by Staphylococcus This is the accumulating form of lugdunin, which acts as an
lugdunensis, which competes with pathogenic Staphylococcus antibiotic against S. aureus strains in the oral cavity.
aureus strains in human oral cavities.[23] The lugdunin NRPS Another cascade reaction set in motion by a redox step
assembly line generates a tethered heptapeptidyl-enzyme with a nicotinamide co-substrate is catalyzed by the S-
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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.
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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.
adenosylhomocysteine (SAH) lyase, the key enzyme in phile to drive subsequent carbocycle formation. Examples of
returning homocysteine carbons to the cellular metabolic this enzymatic strategy include the formation of plant iridoid
pool after SAM has been used for millions of methyl transfers monoterpene scaffolds[25] and also the tricyclic core of
in every cell cycle (Scheme 7). The enzyme is erroneously bacterial polycyclic tetramate macrolactams.[26] Iridoid scaf-
termed a hydrolase because the thioether linkage in SAH is folds, such as in nepetalactone and the secologanin-derived
converted into free homocysteine and adenosine. The thio- moiety in the oncology agent vincristine, arise from geranyl-
ether is stable to hydrolysis. Instead the SAH lyase first diphosphate (GPP) that is processed to 8-oxogeranial (1,8-
oxidizes C-3 of the ribose ring to the ketone while generating geranyl dialdehyde). Hydride conjugate addition from
NADH, which is kept tightly bound in the active site.[24] The NADPH by iridoid synthase sets up the enolate that reacts
value of the alcohol to ketone oxidation is in acidification of intramolecularly through a Michael cyclization to form the
the adjacent C4 OH since the resultant carbanion is now irodial nucleus nepetalactol (Scheme 8 A).[25b] In analogy, the
stabilizable as the enolate anion. This easily accessible ikarugamycin biosynthetic enzyme IkaB similarly transfers
carbanion can be used to eliminate the homocysteine a hydride to the terminal olefin of the nascent product from
moiety with C5 S cleavage to yield the conjugated enone the IkaA polyketide synthase (Scheme 8 B). A proposed 4+2
with a 4,5-exomethylene. This conjugate enone is the electro- cyclization yields the substrate for IkaC and another hydride-
phile for water addition to yield the 3’-ketoadenosine. Then initiated cyclization to yield the fused 5-6-5 core of the
back transfer of the hydride from bound NADH gives the bacterial metabolite ikarugamycin.[26b]
observed product adenosine. In addition to these NAD/NADH-dependent redox
Initiation of nucleophilic cascades can occur through an enzymes that set off cascade reactions, there are several
initial transfer of a hydride from NAD(P)H to an olefin in flavin-dependent enzymes that conduct redox catalysis that
a co-substrate, thereby creating a transient carbon nucleo- uncover cryptic reactivity in nascent products. Three exam-
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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.
ples are presented. The first is the FAD-enzyme Sol5 that shown. Collapse of the tetrahedral adduct generates an acid
converts the exocyclic alcohol group in prosolanapyrone II attached to the C-ring and an initial enediolate from what had
into the pyrone aldehyde in prosolanapyrone III in a fungus been the D-ring. Ketonization yields the hydroxyketone
that causes potato blight (Scheme 9).[27] The aldehyde is now grouping in the product mithramycin, with net oxygenative/
in conjugation with the adjacent olefin and that apparently hydrolytic fragmentation of the d-ring cyclohexenone.[28]
lowers the energy barrier so that it can act as a dienophile The third FAD enzyme, EncM, catalyzes oxidation of C4
towards the terminal diene. The result is an apparent Diels– in the side chain of the advanced polyketide chain tethered to
Alder [4+2] cyclization to the decalin bicycle in solanapyrone. the type II PKS EncC protein in enterocin biosynthesis.[29] A
The second enzyme is an FAD-containing monooxyge- notable aspect of the EncM catalytic mechanism is the
nase that converts the tetracyclic core of glycosylated discovery that it uses a newly identified flavin-N5-oxide as its
premithramycin B in to the tricyclic ring in mithramycin DK oxygen atom transfer catalyst to the 1,3-diketone methyl-
(Scheme 10). The enzyme MtmOIV is a Baeyer–Villiger ene[30] (Scheme 11). This creates a transient 1,2,3-triketo
catalyst, which delivers a nucleophilic oxygen to the d-ring moiety and sets in motion a Favorskii-type rearrangement,
enone with ring expansion to the lactone. The lactone as presumably generating a hydroxycyclopropanone intermedi-
a cyclic ester is labile to water-catalyzed ring opening as ate. That highly strained electrophile can be captured by an
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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.
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describe the use of epoxides as disappearing electrophiles,

particular in the context of cyclic ether formations. Both of
these strategies have been previously reviewed in the context
of biosynthetic cascades.[32]
4.1. Carbocation-Driven Cascade Rearrangements of Terpene

Scaffolds
The cyclization of terpene substrates is synonymous with

cascade biochemistry. The enzymology of monoterpene (C10),
sesquiterpene (C15), diterpene (C20), and triterpene (C30)
scaffold diversification is replete with cascade reactions that
are mediated by carbocation rearrangements and quenched in
given enzyme sites at specific loci and at specific points in the
reaction manifold.[13, 33]
At the C15 level, farnesyl-diphosphate (FPP) is converted
into the tricyclic-5,5,5-alkene in the active site of pentalenene
synthase via at least four cation rearrangement intermediates
during the assembly of the sesquiterpene antibiotic pentale-
nolactone (Scheme 12).[34] Catalysis is initiated as usual by
early dissociation of the C1 OPP carbon–oxygen bond to the
delocalized allyl cation. Capture of the C1 cation by the C11
double-bond terminus yields the E,Z-humulyl cation, which
can then proceed to a tricyclic 5,6,4-cation before rearrange-
ment to the 5,5,5-tricyclic cation. Proton loss creates the
double bond in the pentalenene product framework as
shown.[35]
At the C20 (diterpene) level, the classic building block is
geranylgeranyl-diphosphate (GGPP). It is the direct precur-
sor of the pentacyclic framework of taxadiene, the 6,8,6-
tricyclic diene that is the product of taxadiene synthase
(Scheme 13).[36] In turn, taxadiene is subsequently oxygenated
to introduce eight oxygen atoms to increase solubility and
yield the end product taxol,[37] a potent ligand for tubulin and
an antimitotic antitumor agent of wide clinical utility. The
conversion of linear tetraenoic GGPP to tricyclic taxadiene is
clearly a cascade process, with the first step converting
a bound conformer of GGPP into the C1 allyl cation with
Scheme 8. Reductive strategies to carbocycle formation in plant iridoid capture by the terminal C17 olefinic carbon. That initial
scaffolds (A) and bacterial polycyclic tetramate macrolactams (B). macrocyclic cation undergoes three further rearrangements,
leading to two new C C bonds on the way to releasing
taxadiene.
The most famous of the polyolefin cyclization cascades
intramolecular OH group acting as nucleophile, as shown, to occur in many variants of triterpene cyclases, with the acyclic
create an internal lactone and altered framework connectiv- C30-hexaene squalene as the starting point. The most straight-
ity. Lactonizing release from the acyl carrier protein EncC forward may be the bacterial squalene-hopene cyclase.[38] The
produces the observed deoxyenterocin product. The complete 2,3-double bond is protonated in the enzyme active site to the
pathway has been reconstituted from pure enterocin biosyn- hopanyl cation to start the multistep capture of that cation by
thesis enzymes in vitro to generate the natural product and the remaining five olefins. This creates a pentacyclic tertiary
designed unnatural products.[31] cation that can be quenched in one of two ways. Proton
abstraction yields hopene (Scheme 14 A), while water addi-
tion gives hopanol.
4. Enzymatic Electrophilic Cascades
Two categories of electrophilic cascades are discussed in 4.2. Enzymatic Methylation as Initiator of a Cyclization Cascade
this section. The most extensive occurs in terpene biosyn-
thetic pathways in which carbocation chemistry dominates A variant of controlled induction of an electrophilic
enzymatic reaction sequences. In the second category, we cyclization occurs in the late stages of assembly of the
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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.
prenylated indole teleocidin B, an inhibitor of protein kin- pling of the valyl nitrogen and C4 on the indole moiety.[41] To
ase C (Scheme 15).[39] The precursor of teleocidin B is the go from lyngbyatoxin A to the teleocidin B stereoisomers
indolactam lyngbyatoxin A, itself the causative agent of requires a new C C bond at C6 of the indole ring. Studies by
seaweed dermatitis.[40] The biosynthesis of lyngbyatoxin A is Abe and colleagues[39] revealed that the methyltransferase
understood to involve a two-module NRPS and then a P450 TleD is the enzyme that sets the appropriate chemical cascade
enzyme (thwarted oxygenase; see the section below on in motion. S-adenosylmethionine donates a [CH3+] equivalent
radical cascades), with a mechanism involving radical cou- to the terminal olefin at C25, creating the initial C26 tertiary
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Scheme 11. The EncM flavoenzyme doubly oxygenates a 1,3-diketo moiety of the enzyme-bound poly-b-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.
4.3. Enzymatic Chlorination to Initiate an Electrophilic Cascade
The merochlorin meroterpenoid antibiotics contain

a 1,3,6,8-tetrahydroxynaphthalene core (from a type III poly-
ketide synthase) connected to a rearranged sesquiterpene
chain arising from prenyltransferase activity.[42] The pre-
merochlorin scaffold is then the substrate for a vanadium-
dependent chloroperoxidase that delivers chloronium ions to
the naphthalene ring and also sets in motion an oxidative
dearomatization/terpene cyclization cascade via carbocation
intermediates that lead to the unusual frameworks of mero-
Scheme 12. A sequiterpene cation rearrangement cascade converts the chlorins A–D (Scheme 16).[42]
acyclic C15 farnesyl-diphosphate into the tricyclic-5,5,5- framework of The vanadium-dependent chloroperoxidase Mcl24 deliv-
pentalenene during the biosynthesis of pentalenolactone. ers a “Cl+” equivalent, most likely from a V OCl species via
an enzyme-bound chloramine intermediate, to C2 of the
tetrahydroxynaphthol nucleus as shown in Scheme 16. Trans-
carbocation. Through deuterium labeling, Abe and colleagues fer of a second chloronium ion equivalent to the C1 OH
established that the C25 H migrates to C26 as a hydride, would give the naphthyl hypochlorite transiently, followed by
creating a transient 28 carbocation at C25. Capture of this elimination of that Cl as a chloride ion by intramolecular
cation by C7 of the indole would yield the indicated 5,6-ring participation of the aromatic double bonds, leading to the
spiro cycle with an exocyclic iminium ion. Ring expansion dearomatized cation. Now cyclization by regioselective
through single-bond migration would quench the charge on capture of that cation by the middle double bond of the side
the indole lactam exocyclic nitrogen and give teleocidin B, chain triene yields the new tertiary cation that can be
thus showing that the capture of the C25 carbanion had been quenched by either of two routes. Route a) yields the product
a re face attack. This cascade expands the enzymatic merochlorin A with fused chloro-cyclopentanone, while
repertoire of electrophilic initiation of cyclizations to route b) gives the fused tetrahydrofuran ring of merochlor-
double-bond protonation, C-methylation, and chlorination in B. More recently, Miles et al have shown that merochlor-
and epoxidation (examined in the next two subsections). in X, the putative precursor to merochlorins C and D, can
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Scheme 13. A diterpene rearrangement cascade converts acyclic C20 geranylgeranyl-diphosphate into tricyclic taxadiene.
come from similar chemical logic in the active site of the epoxide by a vitamin B2-dependent squalene epoxidase. This
Mcl24 chloroperoxidase, in this case via route c) through epoxide is then the electrophilic substrate for oxidosqualene
a chloronium-ion assisted a-hydroxyketone rearrangement cyclase, with participation of the p electrons from four of the
(Scheme 16).[43] Thus Mcl24 single-handedly provides the olefins (not five as in squalene-hopene cyclase, presumably
three merochlorin natural product skeletons from the same because of folded conformer differences in the active sites) to
pre-merochlorin precursor. the protosterol cation (Scheme 14 B, see Ref. [13] for a sum-
Total syntheses of both merochlorins A and B have been mary).[47] A cascade of two hydride and two methyl migrations
achieved.[42b, 44] The four-enzyme pathway to merochlorins in occurs before cation quenching and release of tetracyclic
a single test tube has also been achieved,[45] as well as lanosterol. It has been argued that the squalene substrate in
a unifying hypothesis for naphthoquinone-containing mero- the hopene cyclase is an all pre-chair foldamer but is folded as
terpenoid scaffolds.[43, 46] Comparison of the strategic use of a pre-chair-boat-chair conformer in oxidosqualene cyclase.[47]
“Cl+” in this case versus “CH3+” in the teleocidin case above Correspondingly, a chair-chair-chair foldamer for squalene
shows the different tools available in enzymatic sites to epoxide would give the 6,6,6,6,6-pentacyclic scaffold of b-
initiate electrophilic cascade chemistry. amyrin, the most common steroid scaffold in plant metabo-
We will describe a different halogenation strategy in lism. Conversion of the initial dammarenyl cation via
Section 8, in which chlorine radical equivalents are employed a lupanyl cation into the oleanyl cation is followed by
in an enzymatic cascade to cyclindrocyclophane, and compare quenching through proton abstraction by a specifically placed
it to nonenzymatic metathesis synthetic strategy. enzymatic base (Scheme 14 C).[48]
While many of the steroid cyclases terminate the cation-
driven cascade rearrangements at one specific stage and
4.4. Tandem Epoxide Formation and Opening in Enzymatic region by proton abstraction or water addition, there are
Electrophilic Cascades family members that show some promiscuity. The baruol
synthase from the Arabidopsis plant generates baruol at
The more common modes of squalene enzymatic cycliza- nearly 90 % of the flux, with the remaining material distrib-
tion involve prior epoxidation of the 2,3-double bond to the b- uted over 22 minor products, varying in amounts from 0.02–
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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 b-amyrin product is the
predominant mode in plant metabolism.
2.7 % (Scheme 17).[49] This product range proves that a cas- indoline intermediate then undergoes capture at C2 of the
cade of multiple rearranged cationic species exist and can be indole ring by the neighboring olefin, loss of water, and
quenched, perhaps as 22 minor mistakes of timing and oxidative aromatization.[51d]
placement. The actions of PaxM and PaxB illustrate a widely used
Several examples of disappearing-epoxide strategies cascade strategy of tandem enzymes that make and then open
occur at the interface of indole alkaloid and prenyl transfer epoxides as “disappearing electrophiles” in cascade reac-
enzymology. One such example occurs during assembly of the tions.[52] The squalene epoxidase and oxidocyclase pair above
paxilline scaffold. Indole capture of the C20 GGPP yields 3- are such examples. So are Lsd18 and Lsd19 in lasalocid
geranylgeranyl indole (Scheme 18 A). This is the substrate for biosynthesis, but in that case the nucleophiles initiating the
PaxM, which bis-epoxidizes the terminal two of four side- cascades are not p electrons of olefins but instead the
chain olefins. The bis-epoxide is now subject to enzyme- alkoxide forms of internal -OH groups.[53] As shown in
mediated capture by p electrons of the remaining two double Scheme 19, Lsd19 acts on the bis-epoxide generated by Lsd18
bonds by the PaxB enzyme to create the hexacyclic frame- with regio- and stereospecific control to generate two sizes of
work of paspaline,[50] which upon subsequent double oxygen- cyclic ethers: the five-member dihydrofuran and the six-
ation serves as a precursor to the end product paxillin. A member dihydropyran. Although the enzymes remain to be
related strategy is used in assembly of indolosesquiterpenes, discovered for the dinoflagellate polyether toxins, such as the
represented by the pentacyclic family of xiamycin.[51] The 11-fused ether scaffold of brevetoxin B and 13-cyclic ethers in
conversion of farnesylindole into xiamyicin involves three ciguatoxins, it is anticipated that polyepoxide precursors will
kinds of enzymatic oxygenations (Scheme 18 B). The first is be converted in comparable cascades (Scheme 20).[54] Anal-
a prototypic epoxidation of the terminal olefin of farnesyl ogously, Leadlay and colleagues have presented evidence for
indole and electrophilic closure of two carbacyclic rings to MonC involvement as an epoxidase to create a triepoxide
form preindosespene. The second round of three P450- that, on opening by MonB, yields the five cyclic ethers in the
mediated oxygenations converts an exocyclic methyl into polyether antibiotic monensin.[55] The sequence of polyolefin
the carboxylate via an intermediate alcohol and aldehyde, to polyepoxide to fused pyran and furan ring scaffolds in
consuming three molecules of O2. C C bond formation to cyclic polyethers was predicted by Cane, Celmer, and West-
yield the xiamycin scaffold occurs by flavoenzyme action ley,[53b] and further refined by Spencer and colleagues[55b] for
through a cryptic indole ring hydroxylation. The resultant monensin.
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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 a-hydroxyketone rearrangement and a third chlorination
event.
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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.
One final small-molecule example of the strategy of 5. Pericyclases

deploying epoxides as intermediate electrophiles in enzy-
matic cascades is the assembly of the 2,6-dioxa-bicyclo- Pericyclic reactions have a central place in many of the
[3.2.1]octane scaffold of the ATP synthase inhibitor aurover- multistep syntheses of complex natural product scaffolds.
tin E (Scheme 21).[56] The E,E,E-terminal triene of the Nicolaou et al.[3a] and Baran[6] list a variety of 2+2 and 4+2
polyketide precursor is enzymatically isomerized to the cyclizations, and even a 5+2 cyclization, as well as several
E,E,Z geometric isomer that is now a substrate for the variants of 3,3-Claisen and Cope, azaCope, oxyCope rear-
FAD-containing monooxygenase AurC, which epoxidizes two rangements in a multitude of synthetic campaigns. Hetero-
of the three double bonds. AurD is the paired epoxide 4+2 rearrangements have also been used to in biochemical
hydrolase that catalyzes intermolecular regioselective attack pathways for complexity generation in several contexts.
of water on one of the epoxides to create the incipient For some decades, there has been debate as to whether
oxyanion to open the second epoxide and yield the dihydrox- comparable pericyclic reactions occur in metabolism, partic-
yfuran species. This has one double bond and is again ularly in the secondary metabolic pathways where many
a substrate for the epoxidase AurC. That mono-epoxide is complex scaffolds and frameworks are generated. The debate
once again tandemly acted on by AurD, now acting as an has often centered on whether transformations were actually
intramolecular “epoxide hydrolase”, in a 6-endo-tet opening concerted or stepwise. We note 3,3-rearrangements of the
to create the bicyclo-octane scaffold with fused terahydro- Claisen and Cope types below. Over the past decade,
furan and tetrahydropyran rings, reflecting their epoxide a number of purified enzymes have also been assigned as
heritages. The use of epoxides as disappearing electrophiles to 4+2 cyclization catalysts, although it remains to be proven
initiate cascade reactions is well established as part of natures that any one of the purported 4+2 reactions proceed by
biosynthetic tool set. a concerted mechanism (either synchronous or partially
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Scheme 18. Disappearing-epoxide cascade reactions in the enzymatic conversion of 3-prenylindole substrates into paspaline (A) and xiamycin (B).
asynchronous).[57] Certainly, the 4+2 conversions dramatically SpnG, SpnL then cyclizes the remaining diene to the cyclo-
alter framework connectivity in the enzymatic products.[58] pentene ring in what may be a Rauhut–Currier-type of C C
Some of them, we would put in the cascade category. bond formation.[60] The two enzymes SpnF and SpnL thus
In connection with this, we highlight the tandem action of build the constrained 5,6,5-tricyclic core of spinosyn A
the enzymes PyrE3 and PyrE4 in the pathway to pyrroindo- (Scheme 23). Energetic calculations for the SpnF transition
mycin B (Scheme 22).[59] It is not so much that either enzyme states support a [4+2] reaction but also suggest a possible
by itself is a cascade catalyst but rather that tandem action [6+4] route that would need to be followed by a 3,3-Cope
leads to the generation of the pentacyclic core of the natural rearrangement to give the observed product.[60]
product. Furthermore, the two enzymes illustrate the two The Diels–Alder [4+2] cyclization was originally de-
variants of the kinds of 4+2 outcomes that have been scribed in 1928.[61] An oxo-Diels–Alder reaction to give
catalogued to date in enzyme systems. PyrE3 is a “typical” dihydropyran derivatives was reported in 1949.[62] The corre-
decalin-forming [4+2] catalyst. The next enzyme, PyrE4, sponding intramolecular aza-Diels–Alder reaction on an
takes this dialkyl decalin product and makes a spirotetronate imino alkyne was reported in 2009.[63] The biosyntheses of
ring system as the pentacyclic scaffold is constructed through around 80 members of a peptide antibiotic class that has been
the second type of apparent [4+2] cyclization. morphed into products with a central 2,4,6-trithiazolylpyr-
With some degree of analogy, the insecticide molecule idine embedded in a peptide macrocycle have recently been
spinosyn A has a 5,6,5-tricyclic core that is also of pericyclic formulated to involve an aza-Diels–Alder-type reaction
origin. SpnF has been purified to homogeneity and shown to catalyzed by the purified enzyme ThiM (Scheme 24).[64] The
accelerate a slow non-enzymatic cyclization of the macro- total synthesis of thiocillin and several analogues was
cyclic substrate to the central cyclohexene and the right-hand reported more than a decade ago, and aza-Diels–Alder and
cyclopentane rings. Following enzymatic glycosylation by aza-Mannich chemistry was at the heart of the approaches,
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Scheme 21. Three disappearing epoxide intermediates in assembly of

the bicycloctane scaffold of the ATP synthase inhibitor aurovertin.
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 lasalo-
cid A.
Scheme 22. Tandem action of PyrE3 and PyrE4 as catalysts of two

types of Diels–Alder cyclization in the biosynthetic pathway to pyrroin-
domycin.
which means that they in fact resemble biomimetic strategies

(see Ref. [3a] and references therein). The ThiM catalysis
would be a cascade reaction in that the initial hetero-Diels–
Alder product would be the dihydropyridine still containing
the upstream leader peptide. Aromatizing elimination would
free the upstream leader peptidylamide and yield the
trithiazolyl pyridine. This aza-Diels–Alder reaction also
closes the macrocycle at the same time and imposes a non-
planar geometry on the heteroaromatic trithiazolylpyridine
core (Scheme 24), which is essential to set the three-dimen-
sional conformation of the active antibiotic at the binding site
of the 50S bacterial ribosome subunit.[65] Structural insights of
pyridine synthases involved in the biosynthesis of the related
Scheme 20. It is proposed that 11 double bonds are converted into 13 thiopeptides thiomuracin and GE2270A recently revealed the
epoxides as precursors to the fused cyclic ethers in the potent marine molecular logic in folding the peptide precursor and catalyz-
ciguatoxin. ing formation of the unusual aza-cyclic product.[66]
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the fungal enzyme LepI, which generates the insecticidal

leporin C product (Scheme 25).[67] Because leporin C has
a fused dihydropyran ring system, its biosynthetic gene
cluster was examined to evaluate whether a hetero-Diels–
Alder enzyme might be involved. Indeed, the purified LepI
catalyzed loss of water from the indicated substrate, presum-
ably to yield the exocyclic trienyl ketone. A hetero-Diels–
Scheme 23. SpnF and SpnL build the fused 5,6,5-tricyclic core of the
Scheme 25. The enzyme LepI carries out both a conventional [4+2]
insecticidal agent spinosyn through Diels–Alder and Rauhut–Currier
cyclization and an oxa-[4+2] cyclization competitively. The latter
reactions.
reaction yields the main product leporin C. The spiro product from the
conventional [4+2] pathway is then subjected to an enzymatic [3,3]
An example of an oxo-Diels–Alder enzymatic reaction retro-Claisen reaction to rescue the stranded material and convert it
with an additional novel twist has recently been reported for into leporin C.
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.
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Alder reaction would produce the observed leporin C product by female gametes of marine brown algae. Among the
directly. On the other hand, a conventional [4+2]-catalyzed plethora of related algal cycloalkenes is ectocarpene.[70] This
pathway would yield the indicated spirodecalin. Indeed, this C11 cycloheptadiene is thought to arise from oxygenative
was observed as a co-product with leporin C at early time fragmentation of the D5,8,11,14,17 penta-unsaturated C20 fatty
points of substrate conversion. Later, the enzyme utilized this acid primary metabolite by radical-based peroxide chemistry
co-product and converted it all into leporin C. This is most to the indicated two fragments. Isotope labeling studies are
readily formulated as a retro-Claisen rearrangement consistent with this proposed mechanism but there are no
(Scheme 25). This direction for a 3,3 conversion is the first reports on the enzymatic details (Scheme 27). The 3,3-
seen in an enzyme-catalyzed reaction. In summary, LepI rearrangement step would occur from the indicated trienyl
appears to be able to conduct both a conventional [4+2] and cyclopropane pheromone, detected as an unstable intermedi-
a hetero-[4+2] cyclization reaction competitively (ambivalent ate, and that may be a nonenzymatic thermal reaction at room
transition state), and then retrieve the stranded nascent temperature. It certainly qualifies as a biosynthetic cascade
Diels–Alder product through a retro-Claisen reaction in reaction.
a remarkable cascade of pericyclic transformations. A novel
feature of the LepI enzyme is that it requires the cofactor
SAM, not for its methylating capacity, but rather to use the
positive charge of SAM to direct the pericyclic reactions.
The forward 3,3-Claisen rearrangement of ally vinyl
ethers has been known for decades to be the mechanism of
the key enzymatic transformation in aromatic amino acid
biosynthesis in microbes and plants (Scheme 26). Chorismate
Scheme 26. Chorismate mutase catalyzes the only known [3,3]-rear- Scheme 27. Proposed Cope rearrangement in the biosynthesis of the
rangement in primary metabolism of microbes and plants. brown algal feeding deterrent ectocarpene from a transient cyclo-
propane pheromone, which in turn arises from peroxidative fragmenta-
tion of a polyunsaturated fatty acyl peroxide.
mutase converts the metabolite chorismate into prephen-

ate.[68] In turn prephenate is the substrate for two types of There now appears to be compelling evidence for an
aromatizing decarboxylation enzymes. One mediates loss of enzymatic Cope reaction in the biosynthetic route to hapa-
CO2 with elimination of the -OH substituent as the cyclo- lindole and fischerindole cyanobacterial natural product
hexadiene ring aromatizes. The resultant phenylpyruvate is scaffolds (Scheme 28).[71] The first step is prenylation of 3-
then a precursor to l-phenylalanine upon reductive amina- cis-isocyano-indole by geranyl-diphosphate. The resultant
tion. A competing decarboxylase enzyme ejects not the -OH adduct with two carbon chains at C3 of the indole is proposed
but the -H substituent with its electron pair, as a hydride to undergo a Cope rearrangement, not to C4 of the indole, but
equivalent to NAD, generating NADH and the para-hydrox- as shown onto the isocyano side-chain carbon via a boat
yphenylpyruvate precursor to the proteinogenic amino acid l- transition state. The indoline is a good electron sink for an
tyrosine. These are not cascade reactions but do establish that aza-Prins cyclization to the indicated carbocation. Three
catalyzed pericyclic reactions occur in nature. The other modes for quenching the carbenium ion can lead to three
common 3,3-rearrangement, the Cope rearrangement of 1,5- products. Loss of a proton yields the terminal olefin in 12-epi
dienes, has been less clear in enzymatic systems until very hapalindole C. Capture by C4 of the indole ring yields the
recently. Although the Cope rearrangement pathway has distinct tetracyclic framework of 12-epi hapalindole U. Alter-
been suggested as one alternative for the common formation natively, capture by C2 yields the alternate tetracyclic
of 4-prenyltryptophan in the large class of prenylated indole connectivity of 12-epi fischerindole U, all known products in
alkaloids,[69] it has been difficult to prove that mechanistic this series.[72] An X-ray structure of the enzyme forming 12-epi
alternative. hapalindole U has recently been reported.[73]
There is strong presumptive evidence for a Cope rear-
rangement in the biosynthesis of C11 cycloalkene pheromones
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Scheme 28. Hapalindole and fischerindole biosynthesis arise via

a [3,3]-Cope rearrangement followed by an aza Prins reaction.
6. Radical-Based Cascades
Two general enzymatic routes exist to convert unactivated

C H bonds into carbon-centered radicals that can then
participate in radical-based cascades before release of the
products (Scheme 29). The two alternatives operate at the
opposite ends of the spectrum of O2 concentrations. Iron-
based enzymes that reductively activate co-substrate O2 to
generate high-valent oxo-iron species as proximal oxidizing
reagents represent one evolutionary variant.[74]
At the other end of aerobic/anaerobic range are enzymes
that are typically inactivated by ambient O2. These are the
(predicted) hundreds of thousands of open reading frames in
the radical S-adenosylmethionine (SAM) family.[75] This
enzyme class typically has a tightly bound air-sensitive 4Fe/
4S cluster as a one-electron reductant for bound SAM
molecules.
The SAM cofactor usually participates in methyl transfers Scheme 29. Two enzymatic strategies to homolyze unactivated C H
of [CH3+] equivalents in SN2 type transfers to O, N, S, and even bonds in substrates and create transient carbon-centered radicals
nucleophilic C atoms in a wide variety of small molecule and involving oxygenative and nonoxygenative paths. A) Two variants of
macromolecular substrates. An alternate mode of reactivity is iron-based oxygenases, namely heme-containing cytochrome P450s
evinced in the radical SAM enzyme family.[76] As the name and mononuclear nonheme iron enzymes that require co-substrate a-
ketoglutarate, generate high-valent FeV=O and FeIV=O oxidants, respec-
suggests, one-electron transfer from the 4Fe/4S center to
tively, for co-substrate C H bond homolysis. B) The strategy for
coordinated SAM, yields coordinated methionine and the 5’- homolytic cleavage of S-adenosylmethionine (SAM) in radical SAM
adenosyl radical (dAC). This is the proximal abstractor of HC enzymes by one-electron transfer from a 4Fe/4S cluster to generate the
from C H bonds in bound substrates in active sites of this 5’-deoxyadenosyl radical (dAC) as initiator of substrate C H bond
enzyme family to then create carbon-centered radicals in homolysis.
bound co-substrates.
We present the oxygenase families before the radical
SAM enzymes. The fate of the carbon-centered radicals in 6.1. Oxygenative Paths
these two distinct enzyme classes depends on the substrate
frameworks functional groups and alternative modes and There are two kinds of iron-based enzymes with mono-
timing of radical quenching. In accord with the multiple oxygenase activity, distinguished by the coordination sites
turnover roles of catalysts, both classes of enzymes, the iron- around iron, which control the redox potentials and kinetic
oxygenases and the radical SAM enzymes, must be returned reactivities: heme proteins of the cytochrome P450 (Cyt
to starting oxidation states at the end of each catalytic cycle.[77] P430) superfamily and nonheme mononuclear iron of the two
histidine, one carboxylate ligand family (Scheme 29 A). On
reductive fragmentation of O2, Cyt P450s reach an FeV=O
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oxidation state while the nonheme enzymes reach an FeIV=O downstream partner desacetoxycephalosporin-C synthase
oxidation state. Both high-valent oxo-iron species can cleave (DAOCS) in the formation of these major antibiotic classes
unactivated C H bonds in bound substrates homolytically to (Scheme 30).[78, 80] These O2-reducing nonheme iron oxygen-
yield the substrate radical and Fe(IV/III) OH. For a prototypic ases dramatically alter their substrates in cascade reactions.
monoxygenase outcome, transfer of OHC to substrateC yields IPNS converts the acyclic tripeptide aminoadipoyl-l-cys-
hydroxylated product and regenerates either the FeIII-heme teinyl-d-valine into the fused 4,5-warhead of isopenicillin.
or FeII nonheme resting state for the next catalytic cycle. The l-amino group of the aminoadipoyl side chain is next
There have been many dozens of examples of such hydrox- epimerized by an enzyme to penicillin N. Deep dissections of
ylases reported.[78] (See Ref. [13] for examples in natural the mechanism of both IPNS and DAOCS mechanisms have
product biosynthesis). Some of these oxygenases act itera- validated the radical-based cascades.[81]
tively, for example, on substrate methyl groups to carry out For IPNS, the b-lactam ring is formed first and then the
three steps of two-electron oxidations each, to go from fused five-member thiane ring is constructed, without loss of
a methyl group to an alcohol to an aldehyde to a carboxylic the monocyclic lactam intermediate. Carbon-based and
acid. By some criteria, these could be considered cascade sulfur-based odd-electron intermediates have been proposed.
reactions, but they are not the subject of this discussion. DAOCS in turn uses a high-valent FeIV=O intermediate to
Rather, a significant subset of catalysts in these enzyme cleave a C H bond of penicillin N homolytically at one of the
superfamilies never complete the OHC rebound step.[13, 79] An two prochiral CH3 substituents in the 5-member thiane ring.
alternate fate of the carbon-centered radical intervenes, One can formulate the set of rearrangements as proceeding
typically in a rearrangement cascade. The released product through the 3-member episulfide radical that opens to give
has not incorporated an oxygen atom. Both atoms from the the ring expansion, followed by transfer of HC back to FeIII to
co-substrate O2 end up as in H2O molecules. These enzymes, regenerate starting FeII. The final expanded product has a 6-
we term “thwarted oxygenases”: O2 is reductively activated membered sulfur-containing ring with a C=C double bond
and fragmented, high-valent iron-oxo species are formed as fused to the beta lactam—the hallmark of the cephalosporin
strong oxidants, substrate C H bonds are cleaved homolyti- warheads.
cally, but the oxygen transfer step (OHC rebound) is not Many examples of “thwarted oxygenase outcomes” are
completed. found that reflect cascade intramolecular reactions of bound
The most famous of these rerouted oxygenases are substrate radicals that effectively outcompete the intermo-
perhaps isopenicillin N synthase (IPNS) and its immediate lecular transfer of OHC. Among them are transformation of
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 b-lactam antibiotics through a cascade of radicals.
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the acyclic heptapeptide backbones of vanco-

mycin and congeneric nonribosomal peptides
with triple crosslinks connecting side chains 2–
4, 4–6, and 5–7 to generate the cup-shaped
architecture of this cell-wall-targeting antibac-
terial agent (Scheme 31). Three O2-consuming
P450 enzymes, oxyABC, act while the hepta-
peptide is still tethered on the vancomycin
assembly line, at its last carrier protein domain,
to build two aryl-ether bonds (residues 2–4, 4–
6) and one direct C C bond (residues 5–7)
through radical chemistry on the electron-rich
tyrosyl (residues 2 and 6) and hydroxyphenyl-
glycyl (residues 4, 5 and 7) moieties.[82] This is
tandem peptide cascade chemistry, first to
build the tethered heptapeptide backbone
(Scheme 3 A), and then tandem radical
chemistry three times by the tailoring
“pseudo-oxygenases”. It is clear that in these
cases, the oxygen insertion is epiphenomenal
to the generation of the substrate side-chain
carbon radicals.
Moving from peptide scaffolds to poly-
ketide scaffolds, one can likewise see evidence
of enzymatic cascades from thwarted oxygen-
ases. Notable is the enzymatic conversion of
griseophenone B into the 5,6-spirocyclic
system in the antifungal drug griseofulvin
(Scheme 32) by the P450 enzyme GsfF.[83]
Alkaloid biosynthesis also presents
a number of such cases of intramolecular
routing of substrate radicals generated by
high-valent iron-oxo species in biosynthetic
enzyme active sites (Scheme 33). These
include the conversion of R-reticuline into
salutaridine in the morphine biosynthetic path-
way,[84] and the apparent coupling of two
indolyl radicals to form the indolecarbazole
core in staurosporine and rebeccamycin by the
StaP and RebP heme proteins, respectively.[85]
In the prenylated indole alkaloid family of
fumitremorgins, tryprostatin A is converted
into fumitremorgin C by the P450 enzyme
FtmE. O2 is reduced but not incorporated
into the product framework during formation
of the new C N bond.[86]
Among the most spectacular of the cascade
reactions with a thwarted oxygenase P450
enzyme is the generation of the core of the
communesin natural products by CsnC
(Scheme 34).[87] Tryptophan is the precursor
primary metabolite to tryptamine by decar-
boxylation and to aurantioclavine by prenyla-
tion at C4 and cyclization of the allyl cation.[88]
The FeV=O oxidant in the CsnC heme group is Scheme 31. The vancomycin heptapeptide is tailored on the NRPS assembly line by
proposed to cleave the indole N H bond of three dedicated P450 enzymes. Each acts as a thwarted oxygenase, generating carbon-
tryptamine homolytically and generate based radicals in the electron-rich aromatic side chains of the heptapeptide and
a bound tryptamine C3 radical. If a comparable 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).
C3 aurantioclavine radical could also be gen-
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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).
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biosynthesis of the gibberellin diterpenoid phytohormone.[90]

The second are a set of maturations of the anditomin scaffold
by AntA and AntF to create alternate ring connectivity
within the starting pentacyclic framework.[91]
The tetracyclic ent-kaurenoic acid (Scheme 35) undergoes
one hydroxylation at C7 to the 7-a-OH by the plant enzyme
Cyp88A. In the process of a second hydroxylation to the
gemdiol and thereby the aldehyde, a rearrangement inter-
venes, initiated by C6 H cleavage to yield the C6 radical.[90]
This intermediate undergoes ring contraction of the six-
membered B ring to a five-member ring with an extruded
-C6HC OH. This is the rearranged radical that catches the OHC
equivalent on rebound to form the gem diol. Loss of water
gives the ring-contracted gibberellic acid-12-aldehyde. This is
not a thwarted oxygenase but one that pauses between carbon
radical formation and delivery of the OHC equivalent, leaving
time for framework rearrangement. A convergent pathway to
gibberellin has also been characterized in fungi and bacte-
ria.[92] The fungal gibberellin pathway also employs a single
P450 enzyme (P450-1) to catalyze the conversion of ent-
kaurenoic acid into gibberellic acid-12-aldehyde and the more
oxidized gibberellic acid-7 lactone,[92b] while bacteria employ
two P450s and a dehydrogenase to catalyze the conversion to
gibberellic acid-12.[92c]
The anditomin meroterpenoids are highly oxygenated
molecular structures with some novel bridging rings in the
complex scaffolds, including a highly congested bicyclo-
[2.2.2]octane component (Scheme 36). The building blocks
of 3,5-dimethylorsellinic acid and farnesyl-diphosphate are
condensed, epoxidized, and enzymatically cyclized by AndB
to the pentacyclic preandiloid framework. The iron-based
oxygenase AndA then sets off a radical-based framework
rearrangement that alters connectivity, and sets up a fused g-
lactone and a new carbon bridge on the way to the
bicyclooctane unit.[91] The cascade is set in motion by
homolytic cleavage of one of the methyl C12 H bonds by
the FeIV=O active site oxidant. That primary radical is
Scheme 34. The biosynthesis of communesin B involves the thwarted
P450 oxygenase CnsC, which mediates coupling between the trypt-
converted into an exomethylene group as the neighboring
amine and aurantioclavine radical partners. C8 O ether bond is cleaved to generate a 5’ carbon radical
and adjacent ketone. Reaction of the C12 olefin terminus with
that 5’ carbon radical creates the new CH2 bridge with
erated, and the two radicals couple, a 3-3’-dimeric adduct unpaired electron density at C8. Another olefin-radical
would result as nascent product. Imine exchange and double coupling ensues to set the final hexacyclic connectivity
aminal condensation would yield the communesin core. before HC transfer by reducing agent generates product
Subsequent N-methylation, epoxidation, and addition of andiconin, with an embedded bicyclo[2.2.2]octane moiety.
a polyketide hexadienyl acyl group yields communesin B. No oxygenation of the scaffold has occurred. The X-ray
Because of communesins complex core structure, many crystal structure of AndA in complex with its substrates and
syntheses have been established, including a recent biomim- computational calculations have recently provided further
etic enantioselective approach.[89] insight in how AndA initiates this cascade reaction.[91b] The
enzyme AndF, which occurs further along in the pathway
6.1.1. Ring Contraction and Formation by Iron-Enzyme Cascades after the action of a Baeyer–Villigerase, is likewise a mono-
nuclear nonheme oxygenase family member that also enables
The complexity generation reactions that carbon radicals, radical-based nonoxygenative rearrangement outcomes to get
formed in enzyme active sites by high valent oxo-iron species, to anditomin at the end of 12 enzymatic steps in this pathway
participate in include framework rearrangements that can (Scheme 36).[91a]
result in ring contraction and carbon extrusion as well as ring
formation. Two recent examples are presented here. One is
the ring contraction of ent-kaurenoic acid by Cyp88A on the
way to gibberellic acid 12-aldehyde, an intermediate in the
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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 -C6HC(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.
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6.1.2. Okaramine: One Satisfied and Two Thwarted Oxygenases N H bond homolytically (Scheme 37). Rather than complet-
in a Pathway That Builds an Octacyclic Scaffold in Five ing a net N-hydroxylation event, the nitrogen radical reacts
Steps with the nearby terminal olefin to form a new C N bond. This
olefin-radical coupling is analogous to the step noted for
Penicillium molds are famous for their biosynthetic AndA above. In this case, it creates an eight-membered
capacity to build heterocyclic scaffolds, among them of heterocycle, an azocine ring radical that can be reduced by
course the penicillins and cephalosporins. Recently the path- one electron/HC transfer to the azocine.
way to the octacyclic framework of the fungal metabolite Now the third “oxygenase”, OkaE, goes to work
okaramine E, an insect ion-channel blocker, has been shown (Scheme 37). It acts on the other end of the substrate,
to occur in a short efficient five-enzyme pathway from the cleaving the C H bond between the two five-member
primary metabolites l-tryptophan and D2-isopentenyl diphos- pyrroline rings homolytically to generate the indicated
phate (Scheme 37).[93] The first enzyme is a two-module carbon radical. As with the previous enzyme, this carbon-
NRPS assembly line, which condenses two molecules of l- centered radical can react with the neighboring prenyl-group-
tryptophan and mediates intramolecular capture by the amine derived olefin, in this case to form a four-member azetidine
of the second tryptophan residue to release the cyclic ring radical. One electron/HC reduction completes the five-
diketopiperazine. A prenyltransferase then asymmetrically step formation of the octacyclic scaffold of okaramine E, with
prenylates one indole moiety at N1 and the other at C2 with new 4-ring, 5-ring and 8-ring fused heterocycles from three
capture of the allyl cations at their C3 rather than C1 centers to cascades. Oxygen delivery to substrate is kinetically compe-
give “reversed” regiochemistry of prenylation, which is tent in only the first of the three oxygenases and emphasizes
important for subsequent cascades with the last two enzymes the different roles for flavoenzymes versus iron enzyme
in the pathway to okaramine E. These two steps set up the oxygenases.
action of three oxygenases that represent the three known
monoxoygenase types: a flavoenzyme, a P450 heme iron- 6.1.3. Phenylpropanoid Thwarted Oxygenases
containing enzyme, and a mononuclear nonheme iron
enzyme. A final two examples, of many that could be cited (see
The latter two are the “thwarted oxygenases” while the Ref. [13], Chapter 7), come from plant phenylpropanoid
FAD enzyme, OkaB, actually acts to epoxidize the indole natural product pathways. One is the dimerization of coniferyl
moiety that is prenylated at N1. This reaction sets off a short alcohol to a set of three distinct regioisomeric dimers, with
cascade in which the adjacent amide NH of the diketopiper- dramatically different 8-8’ connectivities (Scheme 38 A).[94]
azine, weak nucleophile that it is, opens the epoxide regio- Pinoresinol is the precursor to a variety of downstream
and stereospecifically to create a new cyclopentane ring natural products in plants,[95] including sesamin and podo-
interposed between the indole and the diketopiperazine in phyllotoxin. A second classic case is the enzymatic conversion
a fused tetracyclic array in okaramine C. of flavonoid to isoflavonoid scaffolds in plant secondary
This is the substrate for OkaD, the P450 enzyme. It uses metabolism, as exemplified by the O2- and P450-mediated
the FeV=O oxidant to cleave the remaining diketopiperazine conversion of naringenin into genistein,[96] itself a precursor to
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.
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with a 1,2-aryl group shift among carbon radical intermedi-

ates.
6.2. Nonoxygenative Paths: The 5’-Deoxyadenosyl Radical as

Initial Oxidant of Bound Substrates
A second and independent route to generate substrate

radicals is presumed to have evolved early in anaerobes and
retained for use in anaerobic/microaerophilic microenviron-
ments in aerobic organisms. In lieu of high-valent oxo-iron
species as powerful oxidants in these enzyme active sites, the
common coenzyme S-adenosylmethionine (SAM) undergoes
radical fragmentation on receipt of one electron from
a conserved 4Fe/4S cluster in the active site (Scheme 29 B).
The methionine fragment remains coordinated to the iron-
sulfur cluster for subsequent re-ligation, while the 5’-deoxy-
adenosyl radical (5’dAC) is most often the initiator of
subsequent chemistry on a bound substrate molecule. As
with the high-valent oxo-iron states in the above class of
monooxygenases, the 5’dAC is a strong enough oxidant to
cleave a nearby C H bond, even when unactivated, to create
the methyl group of 5’-deoxyadenosine (5’-dA) and the
resultant substrate radical.[97]
Depending on the substrate, a variety of chemical out-
comes are possible via radical cascades. These include
isomerization of l-(2S)-lysine to beta-lysine,[98] insertion of
a sulfur atom into desthiobiotin,[99] and double sulfur insertion
into octanoyl thioesters to yield the 7,8-dithioctanoyl product
that is the coenzyme lipoic acid.[100] The fate of the SAM
substrate can be regeneration at the end of a catalytic cycle or
net fragmentation to methionine and 5’-deoxyadenosine.
Vitamin K, a menaquinone, has a naphthoquinone
nucleus that functions as a one-electron redox carrier in
bacterial electron transport and plant photosynthetic electron
transport chains as well as mammalian blood coagulation. In
addition to the long known aerobic biosynthetic pathway
from chorismate via isochorismate and ortho-succinylben-
zoate, an anaerobic pathway involving radical chemistry has
recently been discovered.[101] Two radical SAM enzymes,
MqnE and MqnC, process the same starting chorismate
first to aminofutalosine,[102] then to cyclic dehypoxanthine
futalosine[103] on the way to 1,4-dihydroxy-6-naphthoate
(Scheme 39), a remarkable tandem set of radical-based
chemical transformations in cofactor biosynthesis.[104]
In the conversion of the inactive apo forms of microbial
[FeFe]-hydrogenases to the active forms containing both
carbon monoxide and cyanide ion as ligands to active-site
iron-sulfur clusters, the common amino acid tyrosine is
fragmented to para-cresol and the two desired one-carbon
cyanide ion and carbon monoxide molecules (Scheme 40).[105]
Scheme 38. Phenylpropanoid metabolite O2-dependent diverted radical This is a fascinating radical SAM enzyme cascade. About
cascades. A) Oxidative dimerization of coniferyl alcohol to different
a third of the more than 100 000 open reading frames
lignan frameworks. B) Enzymatic flux of flavonoids to isoflavonoids by
1,2-aryl migration in a radical intermediate. predicted to be radical SAM enzymes in protein data bases
are also thought to contain vitamin B12, also a potential
radical generator as well as a methyl donor, thus indicating
many plant defense phytoalexins (Scheme 38 B). The reaction much new radical cascade chemistry to be elucidated.
catalyzed by isoflavone synthase features as a short cascade
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Scheme 39. Two radical SAM enzymes in the anaerobic aminofutalosine pathway to menaquinone (vitamin K).
7. Cylindrocyclophane: Nonenzymatic Metathesis works together. A prominent example noted by Nicolaou et

Versus Apparent Friedel–Crafts Enzymatic Bis- al.[3a] was the approach by Smith and colleagues[106] to employ
alkylation olefin metathesis dimerization -to form the 7,7-para-cyclo-
phane core of the natural product cylindrocyclophane F
One of the mainstays of synthetic chemical strategies to (Scheme 41 A). Although this molecule had been synthesized
complex natural product scaffolds has been the use of in a stepwise manner previously, Smiths group built the
transition-metal-catalyzed olefin metathesis -to stitch frame- indicated resorcinol monomer and exposed it to a Schrock
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of reversible olefin metatheses that settle at the thermody-

namic minimum.
To date, no known biosynthetic analogues of metal-
coordinated olefin metathesis equivalents are known in
enzymatic catalysis. On the other hand, cyanobacteria do
produce cylindrocyclophane F,[107] albeit by a quite different
cascade logic.[108] Balskus and co-workers established that the
primary metabolite decanoic acid is the starting substrate for
tethering by thioester linkage to a 10 kDa acyl carrier
protein.[108] Then a novel member of the diiron-dicarboxylate
enzyme family CylC carries out a regio- and stereospecific
chlorination of the unactivated methylene group at C6 of the
10-carbon acyl chain to yield the 6R-chloro-decanoyl-S-acyl
carrier protein. This is the substrate for a PKS assembly line
that builds and then releases the S-methyl-R-chloroalkyl
resorcinol (Scheme 41 B). The proximal chlorination reagent
is likely to be a chlorine atom [ClC] rather than the [Cl+]
equivalent noted earlier in the merochlorin pathway. The
diiron reagent in the active site likely homolyzes one of the
two prochiral C H bonds at C6 of the decanoyl chain, with
[ClC] rebound. Now, this saturated chloroalkyl chain, not
a metathesis substrate even if such a bioreagent were present,
undergoes stepwise condensation by the Ca2+-dependent
enzyme CylK, which acts as a double Friedel–Crafts alkylat-
Scheme 40. In hydrogenase activation, a radical SAM enzyme supplies
ing agent (Scheme 41 B). The exact mechanism of bond
CO and CN as ligands for the active-site iron through radical
fragmentation of substrate l-tyrosine. formation is yet to be identified but tandem Friedel–Crafts
alkylations to set the para-cyclophane macrocycle is the
default proposal. This cascade is a novel reaction sequence
that has not previously been reported.
molybdenum carbene reagent to effect two metathesis steps,
via the presumed intermediate shown. The E,E-diene-7,7-
cyclophane product is a reduction and deprotection step away 8. Summary and Outlook
from cylindrocyclophane F. Mechanistic analysis suggested
that the dimerization–macrocyclization product was the Cascade reactions in synthetic chemistry, particularly in
thermodynamically favored isomer, arising from a cascade assembly of the complex frameworks of many classes of
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.
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bioactive natural products, have garnered attention and Acknowledgements

admiration for elegant control of developing molecular
architecture. Many synthetic campaigns have cited biomim- We thank Yi Tang (UCLA) for assistance with the prepara-
etic strategies as inspiration, based on known building blocks tion of the images and Stefan Diethelm (UCSD) for insightful
and chemical reactivity considerations, although the constit- discussions that led to the inception of this Review article.
uent enzymes most often had not been identified. B.S.M. acknowledges support of the NIH through grant R01-
The renaissance in natural product biosynthesis, driven by AI047818 for supporting his work on biosynthetic cascade
two decades of genome sequencing, has now dramatically reactions to bioactive natural products.
changed our understanding of biocatalysts. Genome mining
for biosynthetic gene clusters, transfer of the genes to
surrogate hosts for expression of encoded proteins, and Conflict of interest
sensitive mass spectrometry methods have allowed the
investigation of dozens of enzymes that in fact carry out sets The authors declare no conflict of interest.
of cascade reactions as substrates are converted into products
with remarkable complexity generation.
In this Review, we have used the same categories of
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Tetrahedron 1992, 48, 3001 – 3006. Version of record online: && &&, &&&&
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Reviews
Biosynthesis Fantastic four: Generally, enzymes are
highly selective catalysts for single reac-
C. T. Walsh, B. S. Moore* &&&&—&&&& tions. However, some enzymes instead
control a series of reactions in a cascade-
Enzymatic Cascade Reactions in like fashion. This Review highlights four
Biosynthesis types of enzymatic cascade strategies,
mediated by nucleophilic, electrophilic,
pericyclic, and radical-based reactions,
observed in the biosynthesis of complex
natural products
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Enzymatic Cascade RX

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International Edition: DOI: 10.1002/anie.201807844

Enzymatic Cascade Reactions in Biosynthesis

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Enzyme-mediated cascade reactions are widespread in biosynthesis. From the Contents

1. Introduction to Cascade Reactions in Total 8. Summary and Outlook 31

Cascade reactions have attracted special attention both

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Christopher Walsh is a consulting professor Bradley Moore is Professor of Marine Chem-

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describe the use of epoxides as disappearing electrophiles,

4.1. Carbocation-Driven Cascade Rearrangements of Terpene

The cyclization of terpene substrates is synonymous with

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4.3. Enzymatic Chlorination to Initiate an Electrophilic Cascade

The merochlorin meroterpenoid antibiotics contain

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One final small-molecule example of the strategy of 5. Pericyclases

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Scheme 21. Three disappearing epoxide intermediates in assembly of

Scheme 19. Tandem action of the epoxidase Lsd18 and “epoxide

Scheme 22. Tandem action of PyrE3 and PyrE4 as catalysts of two

which means that they in fact resemble biomimetic strategies

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the fungal enzyme LepI, which generates the insecticidal

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mutase converts the metabolite chorismate into prephen-

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Scheme 28. Hapalindole and fischerindole biosynthesis arise via

Two general enzymatic routes exist to convert unactivated

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the acyclic heptapeptide backbones of vanco-

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biosynthesis of the gibberellin diterpenoid phytohormone.[90]

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with a 1,2-aryl group shift among carbon radical intermedi-

6.2. Nonoxygenative Paths: The 5’-Deoxyadenosyl Radical as

A second and independent route to generate substrate

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7. Cylindrocyclophane: Nonenzymatic Metathesis works together. A prominent example noted by Nicolaou et

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of reversible olefin metatheses that settle at the thermody-

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bioactive natural products, have garnered attention and Acknowledgements