\

Phytochemistry\ Vol[ 38\ No[ 5\ pp[ 0368Ð0496\ 0887

Þ 0887 Published by Elsevier Science Ltd[ All rights reserved
Pergamon Printed in Great Britain
9920Ð8311:87:,*see front matter
PII] S9920Ð8311"87#9906!71

REVIEW ARTICLE NUMBER 024

BIOSYNTHESIS IN THE ROTENOID GROUP OF NATURAL

PRODUCTS] APPLICATIONS OF ISOTOPE METHODOLOGY

LESLIE CROMBIE and DONALD A[ WHITING

Department of Chemistry\ University of Nottingham\ Nottingham\ NG6 1RD\ U[K[

"Received in revised form 12 January 0887#

Key Word Index*Derris elliptica^ Amorpha fruticosa^ Tephrosia vo`elii^ Leguminosae^ biosyn!
thesis^ isotope methodology^ natural rotenoids[

Abstract*Using the natural rotenoids rotenone\ amorphigenin\ deguelin\ rotenonic acid\ dalpanol and mundu!
serone as examples\ their phytochemical biosynthesis has been examined in Derris elliptica\ Amorpha fruticosa
and Tephrosia vo`elii[ The rotenoids are advanced iso~avonoids\ and construction of their angular A:B:C:D!
ring systems has been studied experimentally starting out from simple primary metabolites and passing through
a series of mainly oxidative phases[ The oxidative reactions of rot!1?!enonic acid which biosynthetically form
the E!rings of rotenone\ amorphigenin\ dalpanol and deguelin have been studied in both chemical and
stereochemical detail using seedling preparations and the enzyme deguelin cyclase[ This investigation has
extensively employed substrates isotopically labelled with 03C\ 02C\ 1H and 2H and\ particularly in connection
with E!ring biosynthesis\ required new approaches to the demanding chemical and stereochemical requirements
of the experimentation[ Þ 0887 Published by Elsevier Science Ltd[ All rights reserved

INTRODUCTION degradation\ along with spectroscopy[ Whole plant

The {{secondary metabolites|| of plants constitute an
enormous range of chemical structures that require
study from the many di}erent disciplinary per!
spectives which are included within the term {{phy!
tochemistry||[ Spectacular advances in plant genetics
have had a great impact on enzymology but other
aspects of study have progressed more slowly[ Dixon
ð0Ł has drawn attention to this in the comment] {{The
major limitation for utilisation of DNA from gene
banks does not appear to be technology\ but the lack
of detailed basic knowledge concerning the biochem!
istry\ bioactivity and pharmacology of plant prod!
ucts||[
The isotopic labelling of substrates\ and their
employment in experiments at di}ering levels of
sophistication\ plays an important part in the meth!
odology of phytochemical biosynthesis and although
the ideal is their use together with isolated enzymes\
or at least organelle or tissue!culture preparations\
much of the shape of the _eld has resulted from initial
whole plant experiments[ Speci_c isotopic labelling of
candidate organic compounds\ and the exact recog!
nition of the label in their metabolic products\ some!
times by speci_c label extraction\ or spectral means\
is a suitable pursuit for the organic chemist as it
emphasises his traditional skills of synthesis and
feeding experiments have frequently been carried out
by organic chemistry groups\ and Dewick ð1Ł has
recently commented as follows] {{It is a vindication of
the feeding experiment approach that\ as enzymic data
accumulate\ the transformations demonstrated par!
allel those predicted by the feeding experiments||[
This review summarises biosynthetic work on the
rotenoid group of natural products which are best
known in the Leguminosae family "particularly spec!
ies of the genera Derris\ Lonchocarpus\ Milletia\ Neo!
rautanenia and Tephrosia#\ though they are also found
in certain unrelated families such as the Nyctaginaceae
and the monocotyledonous Iridaceae ð1Ð4Ł[ The best
known member of the group is "!#!"5aS\01aS\4?R#!
rotenone 0\ well known as a _sh poison and the major
insecticidal and antifeedant component of the insec!
ticide {{Derris|| obtained commercially from Derris
and Lonchocarpus species ð5Ð00Ł[ Rotenone "or its 5\6!
dihydro!derivative# binds to NADH]ubiquinone
reductase "complex I# of the respiratory electron trans!
port chain and is frequently used in biochemical exper!
imentation ð01Ð05Ł[ The rotenoids have other inter!
esting biological e}ects such as inhibition of the for!
mation of microtubules from tubulin and anti!cancer
activities ð06Ð08Ł[ There is ecological interest in the
in~uence of rotenoids on certain plant:insect relation!

0368
0379 L[ CROMBIE et al[
Fig[ 0[ X!Ray structure of 7?!bromo!"5aS\01aS\4?R#!rotenone[
ships ð19Ł and there is long standing interest in their
use as _sh poisons for restocking waters with more
valuable _sh species ð10Ð12Ł\ or in indigenous _shing[
To be classi_ed structurally as a rotenoid\ a natural
product must contain the heterocyclic core 1\ or be a
structure clearly related to it[ Unexpectedly\ the stable
B:C!fusion is cis!1 because of a destabilising inter!
action between the 0!hydrogen and the 01!carbonyl
in the ~atter trans!isomer 2 ð13Ð17Ł[ The latter unstable
fusion is known synthetically ð18\ 29Ł\ but is not usu!
ally reported in Nature except as blocked to enolis!
ation by a hydroxyl substitution at C!01a ð20\ 21Ł[
The X!ray structure of 4?!bromorotenone ð22Ł "Fig[ 0#
shows the absolute con_guration of the molecule and
its solid state conformation\ which is also the pre!
dominant conformation of cis!rotenoids in solution[
Certain crystal lattices of rotenone itself may\
however\ contain a cis!molecule having an alternative
conformation ð23\ 24Ł[
Plant materials used in earlier biosynthetic work
have been "i# rooted cuttings of Derris elliptica which
produce rotenone 0\ "ii# a germinating seed prep!
aration of Amorpha fruticosa which produces amor!
phigenin "7?!hydroxyrotenone# 3 ð25\ 26Ł#\ and "iii#
germinating seeds of Tephrosia vo`elii which produce
the chromen deguelin 4[ Later studies on the prenyl
residue in deguelin have employed the enzyme\ degue!
lin cyclase\ isolated and puri_ed from the seeds of
Tephrosia vo`elii[ In the work on the rotenoid
A:B:C:D core\ similar experiments were frequently
carried out using both D[ elliptica and A[ fruticosa[
and the results proved to be essentially the same[
ORIGINS OF THE CARBON ATOMS OF ROTENONE AND
AMORPHIGENIN
Scheme 0 shows the overall derivation of the carbon
skeleton from primary metabolites as ascertained by
the administration of speci_cally 03C!labelled precur!
sors[ Phenylalanine samples labelled at a\ b\ and c
were used\ and in each case the ultimate position of
the label in rotenone and amorphigenin was tracked
by degradation ð27\ 28Ł[ An example of the chemical
degradation used for single atom extraction\ carrying
the radioactivity\ is shown in Scheme 1 ð39\ 30Ł[ As
illustrated in Scheme 0 the aryl ring which forms ring!
A has migrated from carbon!c of phenylalanine to
carbon!b in the rotenoids\ thereby suggesting an iso!
~avonoid derivation[ Aromatic ring!D is derived from
the acetate:malonate pathway and the {{extra|| C!5
carbon\ along with the methyl groups of the two ring!
A methoxyls\ will later be shown to be derived from
the methyl group of methionine ð39\ 30Ł[ Inspection
suggests that the E!rings of rotenone and amor!
phigenin derive from prenyl units\ but at an early stage
positive evidence could not be obtained "see later#
as mevalonic lactone and a series of possible prenyl
precursors were not incorporated either by admin!
istration to germinating A[ fruticosa seeds or to wick!
fed D[ elliptica rooted cuttings[
The rotenoids may thus be characterised in deri!
vation as biosynthetically advanced iso~avonoids\
and this agrees with the circumstantial evidence of
their occurrence[ Thus toxicarol 5 and toxicarol iso!
~avone 6 are found together in Derris malaccensis\
ð31Ł whilst dolineone 7 and the iso~avanone 8 co!
occur in Neorautanenia pseudopachyrrhiza ð32\ 33Ł[
The progress of the biosynthesis of rotenoids from
primary metabolites can be divided into a number of
phases as indicated in Fig[ 1 and this sequence\ marked
by increasing oxidative states\ is followed in the ensu!
ing discussion[
THE CHALCONE!FLAVANONE PHASE "Scheme 2#
The conversion of L!phenylalanine into cinnamic
acid\ mediated by the enzyme L!phenylalanine
ammonia lyase "PAL#\ links primary and secondary
metabolism ð34Ð36Ł and is considered to be the regu!
latory step for the chalcone phase of rotenoid biosyn!
thesis[ It involves the enzymic elimination of ammonia
in an antiperiplanar fashion to produce "E#!cinnamic
acid[ Subsequent hydroxylation by cinnamate 3!
hydroxylase "a cytochrome P349 mono!oxygenase
requiring NADPH and O1#\ and the action of 3!coum!
arate CoA ligase\ leads on to 3!coumaric acid as its
CoA derivative 09[ Condensation of the latter with
three acetate units 00 derived from malonyl!CoA\ cat!
alysed by chalcone synthase\ forms the chalcone 01
which is eventually built into the rotenoid structures
via the ~avanone 02[ Chalcone synthase is the _rst
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology 0370

Scheme 0[ Overall derivation of the carbon skeleton of rotenone[

Scheme 1[ Extraction of the 03C!labelled atom from rotenone as 03CO1 via the tertiarybutylperester[

committed enzyme of the multibranched ~a! ~avanone change is mediated by another enzyme\
vonoids:iso~avonoids pathway and is encoded by a chalcone isomerase ð36Ł[
multigene family of at least seven members in soya In Amorpha fruticosa seedlings\ during the for!
bean\ some of which are clustered ð37Ł[ The chalcone! mation of chalcone 01 from a hypothetical polyketide
0371 L[ CROMBIE et al[

Fig[ 1[ Phases in the biosynthesis of rotenoids[

 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology 0372

Scheme 2[ The chalconeÐ~avanone phase[

Scheme 3[ 02CÐ02C Coupling pattern for amorphigenin biosynthetically derived from 02CÐ02C acetate[

03 in which one carbonyl must be reduced "NADPH
dependent reductase# and dehydrated at some stage\
three situations can be distinguished by the use of
acetate labelled with 02C on both carbon atoms
"Scheme 3# "for a collection of 02C NMR data for
rotenoids\ see Ref[ ð38Ł^ for 0H data for rotenoids\ see
Refs ð49Ł and ð40Ł#[ In the _rst of these 04 the folding
is clockwise with the loss of 4?!ðOŁ^ in the second 05
the folding is anticlockwise with loss of 8?!ðOŁ^ and in
06 either 4?!ðOŁ or 8?!ðOŁ is lost after a freely rotating
phloroglucinol unit has been formed[ By using the
INADEQUATE NMR pulse sequence it was shown
that the eventual 02CÐ02C coupling pattern is as in
amorphigenin 07 and hence the mode of folding is as
in chalcone 04 via 08 rather than 05 via 19 ð41\ 42Ł[
Carbonyl reduction and dehydration are assumed to
take place prior to cyclisation\ though this has not
been established^ the cyclisation is a Claisen type of
condensation[ The direction of folding to form the
aromatic ring parallels that found for pisatin 10 ð43\
44Ł[ Rotenoids such as a!toxicarol have a phloro!
glucinol ring!D and in such cases there is no reduction
and dehydration of the original polyketide chain[
Using a cell suspension culture of Pueraria lobata it
0373 L[ CROMBIE et al[
has been shown ð45\ 46Ł that the formation of a chal!
cone having phloroglucinol hydroxylation\ as
opposed to the resorcinol type\ is promoted by chal!
cone synthase in the absence of NADPH\ the res!
orcinol type requiring NADPH[
In the Leguminosae the chalcone 01 "iso!
liquiritigenin# is known to be enzymically converted
into the more thermodynamically stable "1S#!~a!
vanone 02 "liquiritigenin# and the chalcone isomerase
from A[ fruticosa[ has been isolated and puri_ed "150
fold#[ It occurs as two isoenzymes\ requires no co!
factors\ and forms the "1S#!stereoisomer of 02 ð47\
48Ł[ The chalcone isomerase from Phaseolus vul`aris
has also been studied in some detail ð59Ł and that from
Pueraria lobata has been cloned and overexpressed in
Escherichia coli ð50Ł[ Another chalcone!using enzyme
was also discovered during the work on A[ fruticosa
and gave\ as its product\ the novel enol ether chalau!
renol 11 ð47\ 48\ 51Ł[ However\ as we have no evidence
that the latter is directly implicated in the rotenoid
pathway\ it is not discussed further here[
THE FLAVANONE:ISOFLAVONE PHASE "Scheme 4#
This phase of the biosynthesis ð52Ð54Ł involves oxi!
dation\ and the overall 0\1!rearrangement of the aryl
group destined to become ring!A of the rotenoid\ as
mentioned earlier[ Of _ve synthetic ð03C!carbonylŁ
labelled candidate chalcones 12Ð16 administered to A[
fruticosa\ only one\ 12\ was satisfactorily incorporated
into amorphigenin ð53Ł[ Though the other four chal!
cones have hydroxylation:methoxylation patterns
closer to the _nal rotenoid\ it is apparent that the
stages which introduce these features must be delayed
and the entry ~avone in Scheme 4 is liquiritigenin 02[
That the exit compound from Scheme 4 is methylated
is indicated by a comparison of incorporations into
amorphigenin as between the methyl ether 20 "3?!O!
methyldaidzein# and its phenol daidzein 29\ which is
much higher for the former ð53\ 54Ł[
The enzymology of such a ~avanone:iso~avone
rearrangement has been studied by Grisebach and his
colleagues ð55\ 56Ł who have shown that the con!
version of liquiritigenin 02 into daidzein 29 "as well as
naringenin into genistein# is initiated by iso~avone
synthase from elicitor!treated soybean\ an NADPH
dependent P!349 iron!containing microsomal enzyme
ð57Ð69Ł of the endoplasmic reticulum[ The _rst prod!
uct is the hydroxyiso~avanone 17 which is dehydrated
to give the ~avone by a separate soluble enzyme hav!
ing no requirement for NADPH or dioxygen[ San!
kawa et al[ ð60Ł[ have additionally shown\ by using
microsomes from an elicitor treated cell culture of
Pueraria lobata and 07O1\ that the hydroxyl dehy!
drated by the second enzyme contains 07O but there is
no exchange with the 3!carbonyl group such as might
be expected if an enol epoxide mechanism were
involved[
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology 0374

Scheme 4[ The ~avanone:iso~avone phase] enzymes involved[

The mechanism for this important oxidative
rearrangement remains under debate[ Early ideas
based on a direct 0\1!carbonium ion shift "for some
early references to carbonium ion mechanisms\ see
Ref[ ð53Ł# are not now generally held[ More recently
a 0\1!radical shift 21Ð22 has been proposed ð61Ð65Ł[
Though the proposal has the merit of simplicity\ a
di.culty appears to be that the radical involved is
migrating from a more stabilised site to a less stabil!
ised position ð66\ 67Ł[ A dienone intermediate 23 origi!
nally proposed by Pelter et al[ ð68Ł has formed part of
more than one mechanistic proposal but the means
by which it could be formed is controversial[ None!
theless it is perhaps signi_cant that 3?!methoxylated
cinnamic acid destined for iso~avonoid biosynthesis
becomes demethylated even though the hydroxyl at
that position may be remethylated after the iso~avo!
noid rearrangement ð79Ð72Ł[ One suggestion which
accommodates most of the published constraints ð73Ł
is ipso!attack at 3?! as in 24\ or 0?! as in 26\ by the
0375 L[ CROMBIE et al[
P349 enzyme with its activated iron centre in the
electrophilic resonance form Feiii!O¦ postulated for
epoxidation processes[ Feiii!OH is now eliminated
without exchange at the 3!carbonyl or 3?!hydroxyl "cf[
25# to form the dienone ð74Ł[ Since formononetin is
a much more e}ective precursor than daidzein for
amorphigenin in the A[ fruticosa seedling system ð51Ł\
it has been suggested that the dienone in the intact
plant may be decomposed by S!adenosylmethionine
"equivalent to CH2¦ rather than a proton as is found
with the puri_ed synthase#\ giving 18 ð51Ł[ The
developing carbonium ion at C!1 "stabilised in the
transition state by the lone pair of oxygen\ see 27# is
then attacked by the Feiii!OH produced in the earlier
stage "a {{rebound|| mechanism#[ The iso~avone is
now envisaged as being released from the enzyme
leaving the haeme in the Feiii state ready to be reacti!
vated by oxygen after binding another molecule of
substrate[
Rotenoids of the Iridaceae and Nyctaginaceae ð75Ð
78Ł\ lacking the 2?\3?!methoxylation pattern of
rotenone\ are intelligible biosynthetically if they derive
from 1!hydroxycinnamic acid rather than the 3!isomer
and undergo a similar sequence of enzymic changes[
Chemical models "e[g[ the conversion of a ~avanone
to an iso~avone by treatment with thallium trinitrate#
ð89Ł do not appear\ at this stage\ to throw much light
on the biosynthetic mechanism[
It is subsequent to this phase that the pterocarpan
pathway diverges from the rotenoid pathway through
the reduction of the 1\2!double bond[ This is a}ected
by NADPH!iso~avone oxidoreductase\ as studied in
Cicer arietinum[ Because of their phytoalexin charac!
teristics\ pterocarpans and their biosynthesis have
been much investigated "e[g[ ð80Ð85Ł#[
Scheme 5[ The hydroxylation:methoxylation phase[
THE HYDROXYLATION:METHOXYLATION PHASE
"Scheme 5#
As mentioned above ðOC2H2Ł!6!hydroxy!3?!
methoxyiso~avone "cf[ 31# is a good precursor
"9[31)# for amorphigenin in the germinating seed
system of A[ fruticosa\ and labelled 6!hydroxy!1?\3?\4?!
trimethoxyiso~avone "35# is still better "0[76)# ð53Ł[
Between these two intermediates lies a phase in which
the 1?! and 4?! methoxy groups are inserted and the
question of the ordering of these processes is raised[
This was investigated by synthesing a series of _ve
candidate 03C or 2H labelled intermediates and exam!
ining their incorporations in the A[ fruticosa system
ð40Ł[ 2?\6!Dihydroxy!3?!methoxy!ð3!03CŁiso~avone "cf[
32 in Scheme 5# proved to be an excellent "9[86)#
precursor whilst 2?!OC2H2Ł!6!hydroxy!2?\3?!dime!
thoxyiso~avone "30\ cf[ 33# gave a lower but accept!
able incorporation "9[06)#[ The remaining com!
pounds 6!hydroxy!1?\3?!dimethoxyð1!03CŁiso~avone
28\ and 1?\6!dihydroxy!3?!methoxyð1!03CŁiso~avone
39\ gave only low incorporations which indicated that
they were not intermediates on the pathway[ These
results were con_rmed and supplemented by some
competitive experiments ð42Ł[ The iso~avone ð2?!
OC2H2Ł!30 was administered together with ð1!03CŁ!28
when the former was incorporated whilst the latter
was not[ Similarly\ ð2?!OC2H2Ł!30 was matched against
ð1!03CŁ!32 and in this case the incorporations were
similar and they were both accepted as intermediates
on the rotenoid pathway ð42Ł[
As a result of these experiments the proposed major
pathway of hydroxylation:methylation is summarised
as in Scheme 5[ Now that the rearrangement to an
iso~avone has taken place\ ortho!hydroxylation by the
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology 0376

Scheme 6[ Extraction of the atoms labelled by 03C!methionine from rotenone and amorphigenin[

usually accepted arene oxide mechanism ð35\ 36Ł leads
to 2?\6!dihydroxy!3?!methoxyiso~avone 32 which is
then methylated by S!adenosylmethionine to give the
di!ether 33[ 5?!Hydroxylation to 34\ followed by ano!
ther methylation then leads to the tri!ether 35\ an
excellent precursor for amorphigenin[ Both iso!
~avones 32 and 33 are good precursors for amor!
phigenin but it is uncertain whether route A or B is
dominant[ There is room for minor processes opera!
ting on a grid pattern[ One group of natural rotenoids
is based on 1\2!methylenedioxy! rather than 1\2!dime!
thoxy!substitution and it is likely that this departure
takes place on 32 ð86Ł\ or possibly later at the rotenoid
stage\ by a mono!oxygenase P349 mediated radical
reaction[
For comparison\ in the biosynthesis of the iso~avan
isomucronulatol by bladder senna "Colutea arbore!
scens#\ the sequence 2?!hydroxylation\ 1?!hydroxy!
lation\ and then 2?!O!methylation has been proposed
ð87Ł[ From chickpea "Cicer arietinum# Barz and his
colleagues have isolated a microsomal preparation
which carries out 1?! and 2?!hydroxylation of 3?!meth!
oxylated iso~avones "formononetin\ biochanin!A#\
but not\ to any signi_cant extent\ the 3?!hydroxylated
analogues "genistein\ daidzein# ð88\ 099Ł[ The system is
of the NADPH:O1 dependent P349 mono!oxygenase
0377 L[ CROMBIE et al[
Scheme 7[ The rotenoid phase[
class[ An S!adenosyl!L!methionine]~avonoid 6!O!
methyltransferase from the leaves of a Prunus has been
studied ð090Ł\ as has a methyltransferase from Pisum
sativum ð091Ł and from alfalfa "Medica`o sativa# ð092Ł[
THE COMPLETION OF THE ROTENOID A:B:C:D RING
SYSTEM "Scheme 7#
In early work ð27\ 28Ł it was shown by 03C studies
that C!5 of rotenone and amorphigenin\ the so called
{{extra|| carbon\ is derived from methionine[ The
method used for establishing this by degradation\ and
the incorporation relative to that in the methionine
derived methoxyl groups\ is outlined in Scheme 6 ð27\
28Ł[ Incorporation studies using ðOMe!03C or 2H2Ł 1?!
iso~avone 36 also demonstrate\ with excellent incor!
porations\ that the 1?!methoxy carbon becomes the 5!
methylene of amorphigenin or rotenone ð093Ł[ This is
the key step in rotenoid biosynthesis[ Rate of con!
version experiments between iso~avone 36 and the
corresponding iso~avanone 37 show that the latter is
much less acceptable as a precursor!it probably has
to be dehydrogenated _rst ð53\ 54Ł[ The cyclisation
process and its consequences are summarised in
Scheme 7 but there is little knowledge of the enzy!
mology*whether one or more enzymes is involved*
that lies between structure 38 and 49[ It may be repre!
sented as a radical generation and addition process
41Ð42\ with the methyleneoxy radical adding to the
carbonyl conjugated double bond[ It is unclear what
the _rst product of the enzyme reaction is[
That such a proposal is chemically feasible is
demonstrated by the model experiment 47Ð48 ð094\
095Ł[ Here a methyleneoxy radical generated photo!
chemically from 47 adds to the double bond as
expected[ However\ a reductive step is not included in
the chemical demonstration so the product is largely
the 5a\01a!dehydroisorotenone 48\ except for a small
amount of isorotenone formed by hydrogen abstrac!
tion[ Assuming that the rotenoid is to be generated
enzymically at the expected level of oxidation\ the
radical 42 might be reduced by radical abstraction\ or
by donation of another electron and acquisition of a
proton to give 43[ On the other hand\ oxidation leads
to the dehydrorotenoid 44\ and hydrogen transfer
could then follow as a separate step[ Oxidation of the
radical could also give rotenolones 45 "01a!hyd!
roxyrotenoids# directly "these are themselves easily
dehydrated to dehydrorotenoids#[ Detailed enzy!
mology is now needed to throw new light on the situ!
ation[ An alternative ionic process has been suggested
involving electrocyclisation 46\ followed by hydride
donation ð53Ł\ but at the present time this seems less
attractive than a radical mechanism[
"2#!ð5!2H1Ł!8!Demethylmunduserone 51\ syn!
thesised by the vinyl coumaranone 50 route from the
iso~avone 59 as shown in Scheme 8\ is well incor!
porated into amorphigenin by A[ fruticosa seedlings
and the label remains positionally intact ð096Ð009Ł[
The unlabelled compound is viewed as heading the
subset of rotenoids having 1\2!dimethoxy substituents
in ring!A "Scheme 7#[ Similar subsets have 1\2!methy!
lenedioxy\ 3!methoxy or no substitution in ring!A\
whilst additional subsets have an 00!hydroxyl in ring!
D "e[g[ ð000Ł#[ 8!Demethylmunduserone 49 is found in
the Leguminosae as its O!8!methyl ether\ mundu!
serone 40\ obtained from Mundulea sericea ð001Ł[ Thus
methylated\ munduserone may be viewed as a ter!
minal product as it is likely that a free 8!hydroxyl is
required for prenylation at C!7 or C!09[ Prenylation
at C!7 occurs most commonly and the product itself\
5aS\01aS!rot!1?!enonic acid 52 is found as a natural
product in Milletia pachycarpa ð002Ł[ Its presence in
A[ fruticosa can be detected by isotope dilution but
its turnover is rapid ð003Ł[ Usually in a rotenoid the
isoprenyl unit is oxidatively modi_ed as in dalpanol\
rotenone\ amorphigenin\ deguelin\ elliptone\ malaccol
etc[
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology
Scheme 8[ Synthesis of ð5!2HŁ!8!demethylmunduserone[
The evidence available indicates that prenylation
and oxidative modi_cations of the prenyl group are
late stage processes in rotenoid biosynthesis[ Thus
the ð03CŁ!labelled iso~avonoids 53 and 54\ otherwise
structurally suitable as rotenoid precursors\ but hav!
ing fully formed ring!E appendages\ are not incor!
porated into amorphigenin indicating that closure of
ring!B precedes prenylation and elaboration of the
prenyl group ð53Ł[
ROTENOIDS WITH OXIDATIVELY MODIFIED A:B:C:D!
RING SYSTEMS
Whilst de_nitive proof is lacking\ it is believed that
these oxidative changes are generally late!stage pro!
cesses in plants[ This view is indirectly supported by
0378
the studies of the Casida group ð004\ 005Ł on the meta!
bolic degradation of rotenone by insects and
mammals[ However\ there is experimental evidence
that in plants 01a!hydroxyamorphigenin 55 does not
originate solely from hydroxylation of fully formed
amorphigenin when "E#!ð3?!2HŁrot!1?!enonic acid is
administered to A[ fruticosa ð003Ł[ Using rotenone as
an example\ in vitro oxidation at C!01a can give cis!
56 or trans!57 rotenolones as mentioned earlier[ Both
are reasonably stable\ as interconversion by enolis!
ation is blocked[ Similar 01a!hydroxy derivatives\ b!
56 and a!57\ are isolated from plant material in the
case of a number of other rotenoids[ The 5a\01a!dehy!
drorotenoids 58 and analogues of the B!ring lactone
rotenonone 69 are also frequently reported from
Nature[ However\ rotenolones and their 5a\01a!dehy!
0389 L[ CROMBIE et al[
dration products\ as well as rotenonone types[ may
sometimes be artifacts of aerial oxidation "particularly
during work!up or storage# of rotenoids\ so claims for
their identity as natural products of enzyme action
may at times need further veri_cation[ There seems
no doubt that the acetal type 5!hydroxy rotenoids and
their 5a\01a!dehydro!relatives 60\ are genuine natural
products[ An unusual rotenoid 61 having a seven
membered ring!B\ containing two oxygen atoms\ has
been found in Nature ð006Ł\ and the interesting spiro!
rotenoid amorphispironone 62 is probably formed by
enzymic oxidation of deguelin ð007\ 008Ł[ Products of
less deep change such as aromatic hydroxylation\ or
aromatic methylation\ as well as acetylation\ some!
times occur naturally in the rotenoid series[
ISOTOPIC LABELLING AND THE PRENYLATION
PROBLEM IN ROTENOIDS
Biosynthetic stages beyond the formation of the
characteristic A:B:C:D!system demand study of the
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology
prenylation reaction to give 52 "rot!1?!enonic acid#
and its oxidative conversion into the characteristic E!
rings found in the rotenoid "and many other natural
product# series[ La~amme and colleagues ð019Ł have
successfully studied a prenyl transferase in the white
lupin "Lupinus alba# and Grisebach et al[ ð010\ 011Ł
have obtained a microsomal preparation of a prenyl
transferase operative on iso~avonoid phytoalexins in
elicitor challenged soybean cell suspension culture[
Unfortunately incorporations of dimethylallyl pyr!
ophosphate\ mevalonic acid and a whole group of
related test precursors have all proved too low to be
usable in our A[ fruticosa or D[ elliptica systems ð54Ł[
Consequently a di}erent approach was devised[
This involved the use of rotenone or amorphigenin
as easily accessible starting materials and their con!
version\ by reconstructive synthesis ð54Ł\ into iso!
topically labelled materials suitable for biosynthetic
experiments ð012\ 013Ł[ These used the A[ fruticosa or
T[ vo`elii germinating seed systems or a cyclase
enzyme extracted from the seeds of the latter[
ISOTOPIC LABELLING AND RECONSTRUCTIVE
SYNTHESIS
Construction of a set of isotopically labelled tools
is essential to the biological investigation and the
methods of reconstructive synthesis and isotopic
labelling have been extensively employed[ First\
rotenones labelled with 02C or 03C or 1H or 2H at
the methylene group were required and these were
obtained by degradation and re!synthesis as in Scheme
09 ð012Ł[ Under alkaline "but not acid# conditions the
5a\01a!system of rotenone is racemised through b!
elimination ð014\ 015Ł and C!01a was therefore tem!
porarily blocked towards enolisation by enol acetate
formation[ "Alternatively blocking can be as a rot!
enolone derivative made stereospeci_cally via chromic
acid oxidation\ followed by trimethylsilylation]
Scheme 09[ Labelling of rotenone at C!6? with carbon or hydrogen isotope[
0380
deblocking is then later carried out using zinc and
acetic acid ð016Ł#[ The 6?!methylene can now be
removed and replaced by an isotopically labelled
methylene group by using standard Wittig procedures[
Finally\ the 01a!blocking is removed by acid hydroly!
sis of the enol acetate giving ð6?!02CŁ! or ð6?!03CŁ!
rotenone "or a tritiated or deuteriated variant as
desired# retaining the full optical rotation of the orig!
inal rotenone[ A modi_ed procedure ð012Ł can be used
to make methylene!labelled amorphigenin from unla!
belled amorphigenin[
"E#!Labelled 03C! or 02C!rot!1?!enonic acid can now
be made from the labelled rotenone samples by
employing the chemo! and stereo!speci_c reaction
caused by the ether cleaving reagent boron tribromide
as shown in Scheme 00 ð017Ð029Ł[ Ring opening is
followed by reductive debromination with sodium
cyanoborohydride\ which does not reduce the 01!car!
bonyl[ Unlabelled bromorot!1?!enonic acid\ made
similarly\ can be reduced with cyanoboro!deuteride
or !tritide giving "E#!labellin` with ð6?!1HŁ! or ð6?!2HŁ!
ð020Ł[
A quick and easy method of converting 6?!methy!
lene labelled rotenone into "E#!labelled rot!1?!enonic
acid is hydrogenolysis over a Pd:C catalyst in pyridine\
saturated by!product being removed chromato!
graphically[ Unfortunately the hydrogenolysis is only
½74) stereoselective for the "E#!isomer\ though this
is high enough for some types of experiment ð021Ł[
Starting out from amorphigenin\ "Z#!labelling of rot!
1?!enonic acid can be attained by using the alcohol
function of amorphigenin which is _rst brominated to
give compound 70 Scheme 00[ However\ reduction
of the bromide with lithium aluminium deuteride or
tritide was necessary and this not only places a label
at 7? but also reduces the 01!carbonyl which must
therefore be oxidised back to the carbonyl to give ð7?!
1
HŁ! or ð7?!2HŁ!rotenone[ Scheme 01 shows a pro!
cedure for obtaining the latter compounds from
0381 L[ CROMBIE et al[

Scheme 00[ Methods for the isotopic labelling of rot!1?!enonic acid in the E! and in the Z!methyls[

Scheme 01[ Labelling of rotenone at C!7? with isotopic hydrogen using selenium chemistry[

unlabelled rotenone\ rather than from amorphigenin\
using a phenyl selenochloride addition[ Using sodium
cyanoborohydride\ iodo!compound 76 can be con!
verted into ð7?!1HŁ!rotenone and hence to ð7?!1HŁ!rot!
1?!enonic acid\ providing "Z#!labelled material ð012Ł[
Amorphigenin itself\ isotopically labelled at C!7?
can be made from rotenone labelled at C!6? via the
selenium addition compound 78\ using the selenoxide
elimination method shown in Scheme 02 ð012Ł[
The resonances of the 6?!vinyl protons of rotenone
are separate ðd 4[90 and 3[66 in d5 benzeneŁ and it can
be shown by means of the nOe e}ect with the 7?!Me
that the latter signal relates to the "E#!hydrogen atom[
Knowing the identity of the signals\ the isotopically
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology 0382

Scheme 02[ Conversion of ð6?!02C or 03CŁrotenone into ð7?!02C or 02CŁamorphigenin by label transposition[

Scheme 03[ Synthesis of "Z#!ð6?!1HŁ!Rotenone using sulfur chemistry[

labelled "Z#!isomer 84 was prepared as follows
"Scheme 03# ð022Ł[ Using the methodology mentioned
above\ ð6?!1H1Ł!rotenolone was made as its trimethyl!
silyl ether and then treated with benzenesulfenyl
chloride and desilylated to give the anti!Markownikov
adduct 80 which could be isomerised to the thermo!
dynamically stable pair of diastereoisomers 81[ These
were dehydrohalogenated to give a "Z#:"E#!mixture of
geometrical isomers which were separated by HPLC
to produce pure "E#!ð6?!1HŁ!"phenylthio#rotenolone 82
as the major isomer[ This was now desulfurised with
ð0H1Ł!Raney nickel giving 83 and the protecting rot!
enolone hydroxyl was removed by zinc and acetic acid
to a}ord the "Z#!labelled ð6?!1HŁ!rotenone 84[ The "E#!
isomer can be made similarly\ employing ð1H1Ł!Raney
nickel and undeuteriated precursors ð022Ł[
Three di}erent methods have been devised for label!
ling the 5?!pro!S!group of the natural rotenoid
dalpanol\ from Dalber`ia paniculata ð023Ł\ which was
at one time a candidate precursor of rotenone ð024Ł[
One of the methods is shown in Scheme 04 and\ depen!
dent on the type and "Z#!:"E#!isotopic labelling of the
acetylated rotenonic acid starting compound\ is highly
~exible ð025Ł[ It is illustrated with a deuterium label[
The epoxides 87 and 099 are separable by crys!
tallisation and are identi_ed by X!ray analysis] on
deacetylation] one epoxide gives "4?!R\ 5?!S#!"the natu!
ral series# and the other "4?!S\ 5?!R#!dalpanol[
ORIGINS OF THE E!RINGS IN ROTENONE\ DALPANOL
AND AMORPHIGENIN
Because of the success and convenience of the A[
fruticosa seed system which produces amorphigenin
in quantity\ along with rotenone and some dalpanol\
it is of importance to know something of the metabolic
relationships between rotenone\ amorphigenin and
dalpanol[ ð03CŁ!labelled rotenone is an excellent pre!
cursor for amorphigenin\ whilst the labelled diol epi!
mers 096:097 are not "Scheme 05# ð003Ł[ Degradation
0383 L[ CROMBIE et al[

Scheme 04[ Assignment of the prochiral 6?\7?!methyl groups in dalpanol and sterospeci_c labelling of the "pro!S# with
deuterium[

Scheme 05[ Label scrambling during the formation of amorphigenin from rotenone by A[ fruticosa[
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology
Scheme 06[ Hypothesis for the formation of dalpanol and rotenone[
of the amorphigenin produced from rotenone labelled
entirely at the 6?!methylene however shows that there
is not a direct hydroxylation of the methyl group\
but that during the transformation there is a 49]49
scrambling of the original rotenone label between the
vinyl and hydroxymethyl sites of amorphigenin as
in 095[ Because of the possibility of non!biological
scrambling of the label in handling\ considerable care
has been taken to establish that this did not occur
ð003Ł[ The enzymic reaction probably involves for!
mation of a free allylic radical 093\094 with a lifetime
su.cient to permit scrambling before hydroxylation[
Apart from production of amorphigenin by A[ fru!
ticosa germinating seeds there is a rapid turnover of
rot!1?!enonic acid as assessed by isotope dilution with
dalpanol and rotenone being formed ð002Ł[ It thus
seemed desirable to test the validity or otherwise of
the hypothetical pathway summarised in Scheme 06
"E#!ð3?!03CŁ!Rot!1?!enonic acid 098 was administered
and was well incorporated "0[0)# into rotenone[ Fur!
thermore\ the label resided almost entirely in the meth!
ylene of the latter[ Assuming the intervention of the
"1?S[ 2?S#!epoxide 009 and rearside intramolecular
attack\ the product would be "4?R\ 5?S#!dalpanol 000
as required by the known "4?!R#! con_guration of the
0384
latter[ Dehydration would then need to be into the
pro!S!methyl of dalpanol to provide rotenone labelled
in the 6?!methylene[ However\ when "4?R\ 5?S#!ð6?!2HŁ
dalpanol 000 was administered to the seed system
there was no incorporation into either rotenone or
amorphigenin ð012Ł[ Nor was there any incorporation
by wick!fed Derris elliptica cuttings or seedlings of
Tephrosia vo`elii and this hypothesis must be rejected[
Dalpanol is not an intermediate on the pathway to
rotenone[ Nevertheless this may be how dalpanol 000
itself is formed in Nature and hydroxylation at 3?
followed by conversion to a better leaving group 002
may provide the hypothetical precursor of the furan
ring!E rotenoid elliptone 003[ These possibilities have
not been tested experimentally in the rotenoid series
but it has been shown by Tahara et al[ that a cell
free microsomal preparation from the fungus Botrytis
cinerea converts the iso~avone luteone "004\ from
white lupin# into the epoxide 005\ and hence into the
tertiary alcohol 006 which resembles dalpanol ð025Ł
"Scheme 07#[ The enzyme involved is a NADPH!
dependent monooxygenase with the epoxide oxygen
derived from oxygen gas\ but the reaction stereo!
chemistry has not been reported[
A second hypothesis tested was that the biosyn!
0385 L[ CROMBIE et al[

Scheme 07[ E}ect of a microsomal enzyme preparation from B[ cinerea on the iso~avone lutenone[

Scheme 08[ Failure of candidate precursors to be incorporated into rotenone by A[ fruticosa seedlings[

Scheme 19[ Retention of isotopic labelling integrity during the conversion of hydroxyrotenonic acid into amorphigenin[

thesis of rotenone was achieved by an SN1? elimination
from an hydroxylated rot!1?!enonic acid 007 but
experimental test failed to support this too "Scheme
08# ð012Ł[ Nor was the labelled isopropenyl 008 a pre!
cursor[ However when 3?!hydroxyð3?!2HŁrot!1?!enonic
acid 019 was administered to A[ fruticosa good
"0[23)# incorporation into amorphigenin was
attained "Scheme 19# ð012Ł[ Furthermore the tritium
label was shown to be in the hydroxymethyl group
with virtually none in the methylene Scheme 19[ A
similar experiment was then done using 4?!hydroxyð3?!
03
CŁrot!1?!enonic acid 011 and this time\ with good
incorporation\ the label was found by degradation to
be entirely at C!6? of the amorphigenin i[e[ the enzyme
involved is chemoselective for the methyl group rather
than stereoselective[ Both of the labelled hyd!
roxyrotenonic acids used were made by reconstructive
synthesis but both were shown to be present in A[
fruticosa seedlings by the method of isotope dilution
ð012Ł[
These results were con_rmed by administering ð3?!
03
CŁrot!1?!enonic acid 013 with 3?!hydroxyð3?!2HŁrot!
1?!enonic acid 015 in a double labelling experiment
using one and the same batch of seedlings\ and iso!
lating the amorphigenin and the rotenone formed
"Scheme 10#[ Chemical extraction of the radiolabels
from the two products showed that whereas ð3?!2HŁ!
015 is converted into amorphigenin with speci_c label
transfer to C!7?\ there is no detectable transfer into
rotenone[ By contrast ð3?!03CŁ!013 is converted into
rotenone 014 with speci_c label transfer to C!6? of the
latter and into amorphigenin 016:018 with even label
scrambling between C!6? and C!7? ð012Ł[
Competitive administrations to A[ fruticosa of
ð6?03CŁ!rotenone 013 and 3?hydroxyð3?2HŁrot!1?!
enonic acid 015 indicate that the relative importance
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology
Scheme 10[ Chemical and sterochemical relationships in the biosynthesis of rotenone and amorphigenin as demonstrated by
three isotopically labelled precursors and A[ fruticosa seedlings[
of the two pathways to amorphigenin is ½5]0 respec!
tively[ A similar competitive experiment between 4?!
hydroxyð3?!03CŁ! "cf[017# and 3?!hydroxyð3?!2HŁ "015#!
rot!1?!enonic acids indicated that these two com!
pounds were about equally acceptable as precursors
for amorphigenin ð012Ł[
In sum\ the programme of experimentation has elic!
ited the following facts about the formation of the E!
rings in rotenone:amorphigenin]
0[ Apart from producing the rotenoids amor!
phigenin\ rotenone and dalpanol from rot!1?!
enonic acid\ the latter\ as well as the "Z#! and "E#!
forms of hydroxyrot!1?!enonic acid 017 and 015
are found to be present in the germinating seeds of
Amorpha fruticosa[
1[ Rotenone is formed stereospeci_cally from rot!1?!
enonic acid in A[ fruticosa with the "E#!methyl
becoming the methylene of rotenone[
2[ There is no evidence for hydroxylated inter!
mediates which become dehydrated in the for!
mation of rotenone[ It is more probable that an
iron containing monooxygenase is involved within
an overall dehydrogenation process[
3[ Amorphigenin is formed by enzymic hydroxylation
of rotenone\ but in the process a speci_cally pos!
itioned label in rotenone becomes equivalently scat!
0386
tered as between the methylene and the hyd!
roxymethyl of amorphigenin[ It is thus probable that
the intermediate is a resonance stabilised free radical[
4[ Whilst the above hydroxylation of rotenone
accounts for most of the amorphigenin production\
the latter is also formed by cyclisation of either
of the two geometrically isomeric hydroxyrot!1?!
enonic acids 015 and 017[ Isotope dilution con_rms
the presence of both compounds in A[ fruticosa
seedlings[ The production of amorphigenin by the
hydroxylation of rotenone is about 5 times that
from 015 but 015 and 017 are about evenly accept!
able as precursors[
5[ In the conversion of the two hydroxyrot!1?!enonic
acids 015 and 017 into amorphigenin the cyclis!
ation is not stereospeci_c but chemospeci_c for the
methyl group\ be it either "Z#! or "E#![ It is again
probable that resonance stabilised free radicals are
implicated[
Further comments on the mechanism of rotenone for!
mation are made later[
THE ISOLATION OF THE DEGUELIN CYCLASE ENZYME
The plant systems used earlier in this account do not
produce adequate amounts of the dimethylchromen
rotenoid deguelin to allow its formation to be studied\
0387 L[ CROMBIE et al[
Scheme 11[ Failure of a candidate hydroxylated intermediate from rotenonic acid to be converted into deguelin[
and a new germinating seed system from the Leg!
uminous plant Tephrosia vo`elii was developed[ This
produces both deguelin and rotenone and preliminary
experiments showed that labelled rot!1?!enonic acid
was substantially converted into labelled deguelin[
Furthermore it was possible to isolate and purify "782
fold# the water soluble enzyme from ungerminated
seed[ It was named deguelin cyclase ð027\ 028Ł[ The
enzyme does not convert rot!1?!enonic acid into
rotenone\ nor does it convert the "R:S#!hydroxy com!
pound 021 "Scheme 11# into deguelin[ Deguelin
cyclase requires O1 but not co!factors\ though it is
inhibited by chloride ion and by 0\09!phenanthroline
and other chelating agents] iron and copper were
detected[ Although a metallo!enzyme\ it appeared not
to be of the usual P349 type[
On incubating "5aS\01aS#!ð3!03CŁ!rot!1?!enonic acid
with the enzyme there was conversion into deguelin
in theoretical yield\ but using radioactive monitoring
no hydroxylated intermediates could be detected[ It
was concluded that the most likely mechanism for
deguelin formation would be\ in crude mechanistic
terms\ dehydrogenation and a process akin to the
electrocyclisation of a dienone as explained later[
However some subtle stereochemical points arise\ and
these have been studied in some detail ð039\ 030Ł[
Without further quali_cation the two methyl
groups in a 1\1!dimethylchromen are enantiotopic[
When placed in a chiral rotenoid such as deguelin
however the two methyl groups become non!identical
by virtue of the remote chirality at C!5a! and C!01a!
"see 022#[ Each methyl has its own distinctive 0H NMR
signal "dH 0[22 or 0[32# and each its own 02C NMR
signal "dC 17[19 or 17[41#[ The identi_cation and iso!
topic labelling of the "E#! and "Z#!methyls of rot!1!
enonic acid was undertaken as described above and
the problem next to be tackled was to relate each of
the two rot!1?!enonic acid signals to each of the two
deguelin signals[ This was solved as indicated in
Scheme 12 ð039\ 030Ł[
IDENTIFICATION OF THE "pro!R!#! AND "pro!S!#!
CHROMEN METHYL GROUPS OF DEGUELIN
"5aS\01aS#!ð3?!02CŁRot!1?!enonic acid 023 was
treated with phenylselenyl chloride ð031Ł and gave two
"phenylseleno#chromans only\ which were separated
by HPLC[ One "A# showed its labelled methyl at
dC16[62 with the other at 12[14[ The second ster!
eoisomer "B# had its labelled methyl at dC17[38 with
the other at 10[80[ The cyclisation is highly selective
as expected\ with inversion at the selenonium ion giv!
ing the R\S:S\R!products 027 and 039 and not the
R\R:S\S!026 and 028[ The identities of the former
pair were determined by X!ray crystallography which
showed one of them "A# to be 039[ Oxidation to the
selenoxides followed by elimination then gave 030
from 027 and 031 from 039[ The chromen 030 there!
fore has the "5?!pro!S#!methyl resonating at dC17[41
carrying the label whilst 031 must have the "5?!pro!R#!
methyl resonating at dC17[19 ð039\ 030Ł[ The stereo!
chemical study relating to the biosynthesis can now
be undertaken[
THE STEREOCHEMICAL RELATIONSHIP BETWEEN
THE "Z#! AND "E#!METHYL GROUPS OF ROT!1?!
ENONIC ACID AND THE "pro!R#! AND "pro!S#!METHYL
GROUPS OF DEGUELIN CONSEQUENT UPON THE
ENZYMIC CYCLISATION
"5aS\01aS#!ð3?!02CŁRot!1?!enonic acid 033 "i[e[ lab!
elled in the 3?!"E#!methyl# was incubated for 5 h with
an enzyme preparation from T[ vo`elii seeds at pH 6[5
and 14>C when it was completely converted into
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology 0388

Scheme 12[ Identi_cation and isotopic labelling of the "R#! and "S#!methyls of deguelin[

Scheme 13[ Stereochemical relationships between the geometry of the methyl groups of rot!1?!enonic acid and the prochiral
methyl groups of deguelin\ using chemical and enzyme means[

"5aS\01aS#!deguelin[ 02C NMR in the presence of
Cr"acetylacetonate#2 with inverse gated decoupling
showed the 02C!label to be distributed 65) in the "C!
5?!pro!R# and 13) in the "C!5?!pro!S# positions 034
ð039\ 030Ł "Scheme 13#[ It had been expected that the
02
C!label distribution would be either entirely con_ned
to one or other methyl or else distributed evenly[
Indeed a chemical cyclisation using O1 and a pal!
ladium acetate and copper"II# chloride catalyst did
give an even distribution of 02C label between the two
methyls "032#\ the 5a\01a remote chirality having little
in~uence on the ratio ð039\ 030Ł[ The stereochemistry
of the cyclisation was therefore examined in further
detail with respect to the stereochemistry of the C!0?!
H which is removed from rot!1?!enonic acid[
THE STEREOCHEMICAL IDENTIFICATION AND
LABELLING OF THE 0?!"pro!R#! AND THE 0?!"pro!S#!
HYDROGEN ATOMS OF ROT!1?!ENONIC ACID
In order to achieve this\ the "pro!R#:"pro!S# nature
of the 0?!hydrogens of rot!1?!enonic acid had to be
0499 L[ CROMBIE et al[
Scheme 14[ Identi_cation and isotopic labelling of the pro!S!hydrogen of rot!1?!enonic acid[
established\ and a method devised for individually
labelling them ð030\ 032\ 033Ł[
The problem was solved by the creation of the
desired chiral centre on a ring system where control is
more e}ective\ followed by dissolution of the ring
"Scheme 14#[ To this end "5aS\01aS#!deguelin was
treated with benzenesulfenyl chloride to give 035[
Only one stereoisomer was formed and this was con!
verted "using labelled or unlabelled sodium cyano!
borohydride# into 036 with a clean inversion of con!
_guration[ The stereochemistry of 036 was checked
by an X!ray structure determination[ Unfortunately
ring cleavage of the chroman ring by potassium naph!
thalenide did not proceed satisfactorily using 036 but
it did proceed smoothly after reduction of the 01!
carbonyl to form 037[ The 01!carbonyl then had to be
reinstated by oxidation to give the desired 049[ This
procedure gave "5aS\01aS#!ð0?S!2HŁrot!1?!enonic acid
049\ R2H and its deuteriated analogue R1H[
The high stereochemical purity of the 3?\4?!system
in 036 meant that no 3?!epimer was available to be
separated[ The explanation appears to be that the
cationic intermediate is not bridged\ but an {{open||
benzylic cation leading to high syn addition\ and so a
more lengthy procedure had to be adopted "Scheme
15#[ The starting point for the desired labelled "0?!R!#!
rotenonic acid thus became ð3?!2HŁ!deguelin 042 and
this was made from the tritiated intermediate 040 "see
036 in Scheme 14# by sulfoxide elimination 041 : 042[
The labelled deguelin was now taken through a chemi!
cal sequence analogous to that of Scheme 14 to give
the required "0?R#!"5aS\01aS#!ð0?!2HŁrot!1!enonic acid
043 ð030\ 032Ł[
THE STEREOCHEMICAL RELATIONSHIP BETWEEN
THE 0?!"pro!R#! AND 0?!"pro!S#!HYDROGENS OF ROT!
1?!ENONIC ACID AND THE C!5?!"pro!R#! AND C!5?!"pro!S#!
METHYL GROUPS OF DEGUELIN IN THE ENZYMIC
CYCLISATION
Using the pair of rot!1!enonic ð030\ 032Ł acids
stereospeci_cally labelled with tritium in the "0?R#!
046 and "0?S#!044 positions it was shown that within
experimental error there was a correlation between
the removal\ by deguelin cyclase enzyme\ of the "pro!
S!0?#!hydrogen "62)# and the attainment of a "pro!
R!5?#!methyl group "65)# in deguelin 045 "Scheme
16#[ Similarly\ removal of the "pro!R!0?#!hydrogen
"16)# correlates with the attainment of a "pro!S!5?#!
methyl "13)# in deguelin 047[ The stereospeci_c
removal of each of the 0?!hydrogens thus dictates the
direction of rotation of the two methyls attached to
the 3?!position of rot!1?!enonic acid during the for!
mation of deguelin ð030\ 033Ł[
A model for the enzymic cyclisation is suggested in
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology 0490

Scheme 15[ Isotopic labelling of the "0?!pro!R#!hydrogen of rot!1?!enonic acid[

Scheme 16[ Correlation between the removal of the 0?S and 0?R hydrogens of rot!1?!enonic acid by deguelin cyclase and the
attainment of a 5?R and 5?S methyl group in the product deguelin[

Scheme 17[ A hypothetical scheme for the dehydrogenation of rot!1?!enonic acid to deguelin as mediated by deguelin cyclase[

Scheme 18 whilst Scheme 17 contains a suggestion for
the overall reaction stoichiometry[ The model inter!
prets the dehydrogenation as a radical process
mediated by the metallo!enzyme which activates the
oxygen[ As an extreme\ but simple\ representation the
reaction can be viewed as a formation of a dienone by
dehydrogenation and then electrocyclisation involv!
ing clockwise or anticlockwise rotation "see 054 and
055#[ The enzyme requires oxygen but does not require
added co!factors[ No oxygenated intermediates have
been detected and the reaction appears overall to be
a direct dehydrogenation ð030\ 033Ł[
As mentioned above\ we had expected either a
099) stereospeci_c reaction or a 49]49) even dis!
tribution of pathway[ Instead\ a rather {{untidy|| situ!
ation of ½64]14) was found experimentally[
0491 L[ CROMBIE et al[
Scheme 18[ A mechanistic proposal relating the pro!S:pro!R stereochemistry of hydrogen removal in rot!1?enonic acid to
the pro!R:pro!S stereochemistry of the 5?!methyl groups of deguelin[
Assuming that a single enzyme is involved\ it would
appear that the enzyme is not evolutionarily perfected
in a stereochemical sense[ There may indeed be little
evolutionary pressure to attain this[ Our stereo!
chemical awareness of the enzymeÐsubstrate complex
and its subtleties comes only from the use of isotopes
as probes[ From the point of view of the plant\ the
partition between the removal of 0?!HR and 0?!HS is
of no necessary consequence since the starting
material\ rot!1?!enonic acid\ and the cyclised product\
deguelin\ are the same in either case[ Only if the
removal of one particular hydrogen atom by the
enzyme is energetically more e.cient than the other\
might one expect further evolutionary sharpening of
the attack[
LINKS BETWEEN THOSE ENZYMIC CYCLISATIONS OF
ROT!1?!ENONIC ACID WHICH GIVE
DIMETHYLCHROMENS AND THOSE WHICH GIVE
ISOPROPENYLDIHYDROFURANS
Dimethylchromens of the deguelin type occur in
many classes of natural product along with iso!
propenyl dihydrofurans such as occur in rotenone\
and a relationship between the enzymes involved
would not be surprising For example\ a microsomal
preparation from elicitor challenged soybean cell sus!
pension containing P349 type enzyme"s# is reported
to convert the prenylated precursor pterocarpan
glyceollidin 056 into the dimethylchromen glyceollin
II 058 and the isopropenyldihydrofuran glyceollin III
057 "Scheme 29# ð034Ł[ "It does not\ however\ convert
rot!1?!enonic acid into deguelin ð034Ł[# The basic
chemical di}erence between the two modes of cyclisa!
tion appears to be the initial position of the removal
by metallo!enzyme of the hydrogen atom from the
prenyl residue!0?!for dimethylchromen formation
069Ð060\ 3?! for isopropenyldihydrofuran formation
061Ð062 "Scheme 20#[
CONCLUSIONS
The biosynthesis of rotenone\ amorphigenin\ dal!
panol\ deguelin and other rotenoids from primary
metabolites has been established experimentally to
include a series of stages characteristic of those of an
advanced iso~avonoid type[ The individual steps and
the sequence of these and subsequent events leading
to the biosynthesis of the A:B:C:D!ring systems of
rotenoids have been identi_ed by a chemically based
isotopic approach which also throws light on the
classi_cation of rotenoid sub!groups and their oxi!
dation products[ Seedling systems or mature plant
systems have been employed as the biological substra!
tes[ Ring!D prenylation and oxidative modi_cation
of the rot!1?!enonic acid formed leads to the E!ring
systems found in rotenone and amorphigenin[ Isolat!
able oxygenated intermediates involved in these cycli!
 Biosynthesis in the rotenoid group of natural products] applications of isotope methodology 0492

Scheme 29[ Chromen and isopropenyldihydrofuran from a dimethylallyl group in soybean[

Scheme 20[ Comparison of the enzymic dehydrogenative cyclisation of rot!1?!enonic acid to deguelin with that to rotenone
"stereochemistry omitted#[

sations have not been found\ and cyclisations appear
to involve overall direct dehydrogenations[ This is not
the case for dalpanol where an epoxide intermediate
is probable[ The stereochemical e}ects of enzymic
hydroxylations have also been studied[
The isolation of the enzyme deguelin cyclase has
enabled the formation of the dimethylchromen E!ring
of deguelin to be examined in particular ster!
eochemical detail and the mechanism is considered[
During the work on the rotenoid group\ particularly
deguelin\ some rather sophisticated methods of ster!
eochemical isotopic labelling have been developed
which may aid in other biosynthetic contexts where
detailed information is sought[
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