BIOSYNTHESIS OF 7oO-ACETOXYROYLEANONEshodhganga.inflibnet.ac.in/bitstream/10603/33315/10/10_chapter...
Transcript of BIOSYNTHESIS OF 7oO-ACETOXYROYLEANONEshodhganga.inflibnet.ac.in/bitstream/10603/33315/10/10_chapter...
CHAPTER - IV
BIOSYNTHESIS OF 7oO-ACETOXYROYLEANONE
Plant products can be divided into two major groups,
primary and secondary. Polysaccharides, proteins, fats and
nucleic acids which are the fundamental building blocks of
living matter constitute the primary metabolites and are found
in all living organisms° Primary metabolism is the process
which gives rise to these essential metabolites.
On the other hand, there are some chemical processes
restricted to certain species yielding products which do not
play a vital role in the existence of the organism. These are
termed as secondary metabolites and are commonly referred to as
natural products.
Though these compounds possess no obvious metabolic
functions, still they are useful to the plant to some extent.
Some of the secondary metabolites like alkaloids from Atropa.
Berberia. Cinchona. Colchicmn. Ephedra. Holarrhena. Mucuna,
Opium, Kauwolfia. Solanurn. Vinca etc., and steroidal glycosides
(digitoxin derivatives, Ouabain etc.) are of medicinal importance.
Compounds like pyrethrins and nicotine have found their use as
insecticides. Essential oils are used in perfumary industry
and some also in medicine.
The interrelationship between the primary and secondary
metabolism is given in scheme 401<.
1
IV.1 INTRODUCTION TO BIOSYNTHESIS
156
-*► C a r b o h y d r a t e s
C a r b o n d i o x i d e+
W a te r
Iff
(A
Oo>>
O
A r o m a t i c c o m p o u n d *
P y r u v i c a c id
f a t t y a c id sAcetate
P o l y k e t i d e s
A r o m a t i c c o m p o u n d s
A m m o n i a
\\ i
M a l o n a t e
T r i c a r b o x y l i cA c e t y l C o A ----------------- '— + ■ A m in o acids
acid cycle
I0)
a> o c)
a<=o *
x :<u a
♦*Ho<
>a)
£
Ua.
Pro eins A l k a l o i d s
Nucleic acids
T e r p e n o i d s
S t e r o i d s
scheme ; 4.1
157
The distinction between various products is based on
their biogenesis. The term refers to the hypothesis postulated
to describe the formation of natural products based on the
study of compounds possessing a common skeleton and similar
functional group pattern. These underlying common features can
be used to deduce a reaction scheme by which the entire group
can be visualised to have arisen in vivo. The oxidised sites,
however, could arise by processes which are unrelated to the
main biogenetic pathways,, Hence, it is essential that, prior to
deriving any conclusions from the functional groap patterns, a
thorough study of a large number of natural products should be
madec In fact, the validity of any biogenetic scheme is directly
related to the number of compounds whose structures can be
deduced in this wayo It is striking to note that the organism
resorts to the shortest possible path to produce these natural
productso
Reactions which have proved successful in vitro could be
safely assumed to be feasible in vivo too. Thus, with the help
of appropriate enzymes and under physiological conditions, the
organism builds complex molecules from readily available simple
precursors. Often more than one hypothesis is suggested for the
biogenesis of a molecule. The validity of the hypothesis could
be examined by experimental evidences. The actual pathway thus
proposed with the support of experimental inferences is called
'biosynthesis' of the molecule in question.
153
Use of tracers:
The study of biosynthesis of natural products can be
made by the isotopic tracer technique® Till recently, most of
the work involved use of precursors containing radioactive
1 i 3 13carbon ( C) or tritium ( H). Since tbe development of C
nuclear magnetic resonance (CMR) spectroscopy, the stable
13 18nuclide C has featured prominently, while oxygen ( 0) and
15nitrogen ( N) tracers have also been used. The radiochemicals
used can be generally, uniformly, specifically or doubly
labelled. The specific incorporation of the intact substrate
into the product could be determined by the use of doubly
labelled substrate. For obtaining unambiguous results, care
must be taken to ensure radiochemical and chemical purity of
these labelled precursors. The chemically pure labelled natural
product is then degraded to identifiable fragments in which the
position of the label is ascertained. Thus, the metabolic route
leading to the formation of the substance under consideration
can be unambiguously determined.
Plants are complex multicellular organisms which undergo
growth and development accompanied by physiologic specialisation
during their entire life time. Depending upon the specific
function, the biochemical activity of a cell in one organ of the
organism may differ from the activities of cells in other organs
of the same organism<> In terms oi the secondary metabolism, it
seems that at a certain time based upon the growth stage attained
by the plant as well as seasonal variation, the synthesis, or
even a step in the synthesis, of a particular substance may
take place in one part and be elaborated at a later time in
another part of the same planto Consequently, study of such a
synthesis requires examination of a suitable part of the plant
at an appropriate time<> In most cases the problem of selecting
the correct site may be bypassed by using the whole plant. To
choose the proper time is rather difficulto It is known,
however, that the regions of active growth are also the most
active centres of biosynthesis in plants0 Therefore, one may
expect high biosynthetic activity during the active growth
period, ioe0, flowering and fruiting time®
Several techniques have been employed for administration
of labelled precursors to whole plants. In some studies a plant
growing hydroponically is used, and the labelled compound is
added to the aerated nutrient medium. One cannot assume that
the nutrient solution or the plant roots growing therein are
free of micro-organisms» Then there is always a possibility
that the labelled compound is modified or degraded microbially
before it is absorbed by the roots*. To eliminate this error, the
roots are washed with a germicidal solution before immersing them
in the solution of the labelled compound. The disadvantage in
this method is that there is a large dilution of the label. To
overcome this, use of high specil'ic activity precursors becomes
1 6 ( 1
Gases such as CO ̂ are administered in sealed polythene
bags, or in growth chambers, while solid substrates are dissolved
in water or emulsified wi th the help of surface active agents.
The cut ends of the explants can then be dipped into this
solution which is rapidly sucked up by the explant because of
transpiration through the leaves<> Alternatively, the solution
can be either injected into the stem or seed pod by means of
hypodermic syringe or painted onto the leaves. These methods,
being quite tedious and non-quantitative, have been superceded
by the ‘wick technique*. This is a variation of the injection
method and is employed especially in the case of plants having a
strong but not too woody stalko A cotton wick thread is passed
through the stem with the help of a fine sewing needle. The ends
of the two threads are then kept immersed in the tracer solution
contained in a small beakero The solution diffuses slowly into
the plant by capillary action of the thread. After the complete
absorption of the precursor solution within a few hours, the
container is washed with distilled water to ensure maximum
administration of the precursor. The ‘wick technique* has many
advantages over the other methods. The absorption being slow, it
does not disrupt the physiological conditions of the plant to any
great extent. It does not involve large dilutions of the
precursor solution and hence greater amount of the precursor can
be administered in a shorter period of time. Moreover, the method
is quite easy, quantitative and does not need strict supervision.
1 6 1
Alternatively, the biosynthetic sequence of a secondary
metabolite can also be studied using cell free extracts or crude
enzyme systems, auxotrophic methods and plant tissue cultures,,
Ma.jor biogenetic pathways:
The three major biogenetic routes to plant products are
the acetate-malonate, acetate-mevalonate and shikimic acid
pathways.
Acetate-malonate pathway:
The main products of this pathway are the fatty acids
and polyketides. Often, these are collectively referred to as
1acetogenina'. Acetyl coenzyme A (l), the precursor of this
pathway, undergoes carboxylation with carbon dioxide to produce
malonyl coenzyme A (2) with a more reactive methylene group.
This can then react with a second molecule of acetyl coenzyme A
and then decarboxylate to form a four carbon chain, acetoacetyl
coenzyme A (3). The latter can further react with another
malonyl unit in a similar way to provide further linear extension
of tbe polyketide chain which may then cyclise via two major
routes, path ’ a1 and ' b*, to yield two types of phenol derivatives,
v iz ., the acylphloroglucinols (4 ) and the orsellinic acids ( 5 )
(Scheme 4 .2 ). Resorcinol (6) and salicyclic acid (7) derivatives
would be obtained if reduction of an uninvolved ketone occurred
prior to cyclisation. In all probability, the polyketide chain
being highly reactive would remain enzyme bound throughout its
formation and subsequent cyclisationo
1 6 2
CH2COSCoA -f c o 2
( 1 )
CH3CH2CH2COSoAH
HOOCCH2COSCoA
( 2 )
t- CH 3C0 SC 0A
[ ~ c o 2 ,~ C o A S H ]
r
CH 3COCH2COSCoA
( 3 )
CH3(CH2)2nCOOH
Fatty acid
C H 3CO(CH2CO)n CH2COSCoA
Polyketlde
H i CH2CO ) V Y ^ c 00
0 0 0H
H lC H 2C O )n H2C ~ C
OH
s ^ ^
0
HlCH2C0 )n
H O '' "'OH
4)
OH
H ( CH2C0 ) n
OH
( COCH 2 )n H
COOH
<C0CH2)n H
COOH
H O ^ N 0H
I 5)
I C 0 C H 2)n H
COOH
•OH
7 )
scheme : 4.2
163
Alternatively, acetyl CoA gives rise to the aliphatic
amino acids, like ornithine and lysine via the tricarboxylic
acid cycle0
Acetate - mevalonate pathway:
Terpenoids and steroids are biosynthesised by the
acetate - mevalonate pathway. The biogenesis of these compounds
follows the 'Isoprene Buie* according to which they are built up
from isoprene units joined by a head to tail linkages The primary
precursor is mevalonic acid (9), which in turn is derived from
acetyl CoA (l) through the intermediate formation of acetoacetyl
CoA (3) and 3-hydroxy-3~methyl-glutaryl CoA (8) -(Scheme 4 03).
The main difference between the acetate - malonate and
this pathway is the addition of the third acetate unit to
acetoacetyl C0 A0 In the former pathway, as shown earlier, the
linkage is in a linear fashion, while in the latter pathway,
the third acetate molecule is added to the central carbonyl
function to yield a branched six carbon unit, viz., 3-hydroxy-3-
methyi glutaryl CoA (8). The subsequent reduction of (8) gives
mevalonic acid (9 ) which serves as the precursor for various
terpenoidal and steroidal molecules us shown in scheme 4 .4 .
Shikimic acid pathway;
Several important benzenoid compounds in plants are bio
synthesised by shikimic acid pathway also. Indeed, it has been
shown that a large number of aromatic compounds in higher plants
are derived from phenylalanine, tyrosine and tryptophan which
164
2 C H 5 C O S C o A
( 1 )
-► C H 3C O C H 2CO SC o A
( 3 )
. / HO OCC H ^C CH >CH
OHN A D P H + H
-Ml-----■----
V l -v 1'' ,'i. »\*>,►
CHi
H O O C C H 2CCH2C O SC o A
I
OH
( e >
H O O C C H 2 CGH2 CH2OH
OH
( 9 )
scheme : 4..3
M e v a lo n ic acid
( 9 )
D i m e t h y l a l l y l p y roph ospha te
H e m ite rp e n o id s , C 5
11
G e r a n y l p y r o p h o s p h a t e ---------- ► M o n o t e r p e n o i d s , C^q
I r
F a r n e s y l p y r o p h o s p h a t e ---------- ► S e s q u i t e r p e n o d s , < ^ 5
------- ---------------------► S q u a l e n e
1S t e r o i d s T r i t e r p e n o id * f C 3 0
I I
Gferanylgeranyl pyrophosphate — —— “ ®*Ditarp©noid$ , C2 0
i ..P o l y i s o p r e n e s C o r o te n o id s , C 4 Q
scheme:4.4
are the end products of this pathway.
Erythrose-4-phosphate (10) and phosphophenol pyruvate
(ll ), which are the glycolytic products of glucose-6-phosphate,
condense and cyclise to give shikimic acid (12) via quinic and
dehydroquinic acids as shown in scheme 4050 Another molecule
of enol pyruvate adds on to shikimic acid-5-phosphate (13) to
give chorismic acid (14)0 The latter acid is a key intermediate
in the shikimic acid pathwayo It gives rise to tryptophan (16)
via anthranilic acid (l5)» On the other band, it can rearrange
to give prephenic acid (17)<. Phenylalanine (21) and tyrosine
(20) are then obtained from prephenic acid (17) by independent
pathways via phenyl - pyruvic acid (19) and its 4-hydroxyderi-
vative (13) respectively (Scheme 4 <, 5 ) <, It is interesting to
note that the conversion of phenylalanine to tyrosine is rarely
observed in dicot plants^o
Phenylalanine is the precursor for cinnamic acid (22)
and its derivatives like caffeic (23), ferulic (24) and sinapic
(25) acids, the corresponding cinnamyl alcohols and benzoic
acid derivatives (Scheme 406 )0 These phenyl - propanes,
generally denoted as C^-C ̂ compounds, are important intermediates
in the biosynthesis of lignins, lignans, neolignans, flavolignans,
coumarins, flavonoids and a few alkaloids.
167
C H OIC H O H
IC H O H - f
c h 2o H 3
I 10)
C OO H
C H 2
11 m c o [f ]I
C OO H
in)
OEjo^^^'OH
o h
I 13)
IC O O H
CH2
( D o 0 ' ^ N' C O O H
OH
C O O H
0=1B ochT ^ i
h.0' ^ y ^ sn(
OH
COOH C O O H
HOv^COOH
0 " Y ' ' O H H 0 " " ' ' O H
OH O H
HO' - . ^ - C O O H H O
HO
C O O H
C O O H
C O O H
H O ’
O H
H O O C v ^ C H 2 CO C O OH
C H
.A .'O-^vCOOH
I I S ) °XH «U)
\ C O O H
O H
C O O H
HO
XXH
C O O HI
OH
c h 2o[EJHO
/ • ” * X
C H 2 C O C O O H
n h 2
j < ^ V h o > n — c h - c h c h 2o { p ]
o h Ah
IC H — C H C H .0 [El
( I B ) i C H 2C H C O O H
■ II N H 2
O H
( 2 0 )
C H 2 C O C O O H
19) j
C H 2C H C O O H
■ I n h 2
O H O H
SC HEM E.4 . 5
f(21 )
C H 2 C H C O O H
n h 2
(16)
1 6 8
C T "
COOH
{ 22 )
I 2 3 )
COOH COOH
25
SCHEME : 4 .6
IV.2 BIOGENESIS OF TERPENES
Tbe fundamenta1 knowledge of terpene chemistry is due
2 3to the work of Wallach and Buzicka which led to the proposal
of "isoprene hypothesis"., Their original idea is, however,
not correct for it is not free isoprene which is the ultimate
precursor of the terpenes0 The search for the true isoprenvl
precursor occupied a number of different research groups for
many years and several C compounds have been proposed andD
4subsequently discarded . The problem was finally solved by
5 6groups associated with Bloch and Lynen who elucidated the
structure of the precursor and its mode of biosynthesis.
The search for a ’ biological isoprene unit’ met with success
7in the discovery of mevalonic acid which has now been recog
nised as the key metabolite in the terpenoid biosynthesis. It
is also well established how mevalonic acid is formed in vivo
and then transformed into precursors of various classes of
4. 8 » 9terpenes ’ „
Initially two molecules of acetyl coenzyme A (26)
condense to give acetoacetyl coenzyme A (27). The coadensati on
of another molecule of (26) with (27) results in the formation
of p -hydroxy-^-methylglutaryl coenzyme A (28) which is reduced
virtually irreversibly to mevalonic acid (29) by nicotinamide-
adenine dinucleotide phosphate (NADPH). The conversion of
mevalonic acid into biological isoprene unit requires the
presence of adenosine triphosphate (ATP)„ The initial steps
involved are the phosphorylation to give the monophosphate (30)
and then the pyrophosphate (3l) which probably forms the inter
mediate (32). The dehydration and decarboxylation of the latter
gives isopentenyl pyrophosphate (33). Isopenteny] pyrophosphate
(33) is then transformed _in vivo into its isomer, dimethy 1 ally 1
pyrophosphate (34)0 Condensation of (33) with (34), eliminating
a pyrophosphate grouping, results in the formation of geranyl
pyrophosphate (3o), the precursor for monoterpenes<> Condensation
of (35) with another molecule of isopenteny! pyrophosphate (33)
in a similar way gives farnesyl pyrophosphate (36), the precursor
for sesquiterpenes, which can further condense with another
molecule of (33) to generate geranyl pyrophosphate (37), the
precursor for diterpenes<, Squalene, the precursor of the triter
penoids is formed by tail-to-tail reductive condensation of two
molecules of farnesyl pyrophosphate (36) while the carotenoids
are derived similarly from geranylgeranyl pyrophosphate,, All
these transformations are controlled by various enzymes,, The above
steps are schematically presented in scheme 407.
Monoterpenes:
These include acyclic, monocyclic, bicyclic and tricyclic
types. A large percentage occurs in higher plants as hydrocarbons
but alcohols, aldehydes, ketones, acid lactones, oxides, peroxides
have also been found in nature.
The biogenesis of monoterpenes is described in scheme 4<,8
and summarised below:
170
171
C H 3C O S C 0 A +
( 26 )
C H 3C O C H 2C O S C o A
I 27)
CH3
-*■ .c:
h 2c
HOOG
.OH
CM, h 2c
c h 3 .OH
> <CH,
CHj OH
h 2c
(29)
c h 2o h hooc c h 2o p h o o c
130) 131)
'CH-
CH20PP
CH,
H 2C '
C
0 = C
r 1H - 0
OP
CH2
c h 2o p p
CH-,
)HjC
CH2— C H j— OPP
( 33 )
132)
PPO'
(34)
TRITERPENOIDS'*-+ 136)
OPPMONOTERPENOIDS
SESQUITERPENOIDS
OPP(36)
+ 137)CAROTENOIDS * ---- OPP
-DITERPENOIDS
( 37)
SCHEME; 4.7 . B I O G E N E S I S OF T E R P E N O I D S .
CH3COOHHOOC OPP CHO
OH ( 38]
( 40 )
©
©
1 M
9
(42) (43) (44)
\> -----OPP
CHO(47)
S C H E M E . 4 .8 . B I O G E N E S I S OF M O N O T E R P E N E S
i ) Acyclic, Cog«, Citronellal^ (38), Geraniol (39),
nexol (40), linalool** (41)»
ii) Cyclic - Geranyl pyrophosphate can undergo cycli-
12 13sation ’ to give a variety of terpenes like
limonene (42),oc -terpinene (43), oC -phellandrene (44)o
iii ) Non head-to-tail condensation. Artemesia ketone^ (15)
15and chrysanthemum carboxylic acid (46) are examples
of irregular terpenes in which the isoprene units are
not joined in head-to-tail fashion, unlike most of
the terpenes.
iv) Cyclopentanoid, e0g. isoiridomyrmeein^ (47)»
Sesqui terpenes;
Sesquiterpenes occur mostly in higher plants and are
rarely found in lower plants and animal kingdom (e .g ., insects)o
More than four thousand terpenoidal compounds are known so far
and sesquiterpenes numbering around 1200 represent the largest
single classo The recent advances in sesquiterpene chemistry
pertain not merely to their isolation and characterisation but
also include a better understanding of the stereochemistry of
the molecules, their reactivity and rearrangements and also thei
biogenesis, biosynthesis and synthesis.
Structurally, sesquiterpenes possess a cyclic, mono-, bi
tri- and tetracyclic skeleton,. The classification of sesquiter
penes based on Ruzicka's original biogenetic isoprene rule was
• - - 17m i tially due to Hendrickson « This was later extended by
1 7 4
IBParker et alo in 1967 in the comprehensive review of the
biogenesis of sesquiterpenes. Consequently, the carbon skeleton
of virtually all the sesquiterpenes could be derived by suitable
cyclisation of cis-trans farnesyl pyrophosphate (48) and trans-
trans farnesyl pyrophosphate (49)« It is presumed that nero-
lidyl pyrophosphate (50) could also serve as a crucial building
block o
Kemoval of pyrophosphate gronp from (48) yields cations
(51) and (52), and its removal from (49) the cation (53). These
cations (51), (52) and (53) f urther cyclise to give two primary
cations each. Thus the cations (54) and (55) are formed from
(5l), ( 0 6 ) and (57) from (52), and (58) and (59) from (53).
The primary cations from (54-59) embody sesquiterpenoid structural
types directly or by processes involving 1 , 2 or 1,3-hydride shifts,
electronically and sterically controlled cyclisations with the
two remaining double bonds (Markownikoff and anti-Markownikoff' s
cyclisations, Wagner-Meerwein rearrangements) and t,2-methyl
shiftso Based on these, sesquiterpenes can be generally classi
fied into groups such as farnesanes, bicyclofarnesols, bisabolenes,
daucanes, cadinanes, humulanes and caryophy1 1 anes, germacranes,
eudesmanes, aremophilanes, aristolanes and aromadendranes etc.
(Scheme 4 0 9 )o These classes have been extensively revi ewed^ ^ „
Piterpenoids;
Biterpenoids contain four isopentane units and can occur
with a number of structural variations (Scheme 4o10)„ Monocyclic
175
1 76
P h y t o l
I 63)
A g a t h i c a c i d
A b i c t i c a c i d
M a n o o l S c l a r e o l
( 64 )
D e x t r a p i m a r ic a c i d
165)
L e v o p i m a r i c a c i d
P h y I l o c l a d e ne
OH
I s o p h y l l o c l a d e n *
OH
K a h w e o lC a l e s t o l
SCHEME:4.10 p r o m i n e n t m e m b e r s o f t h e d i t e r p e n e
( c 20) FAMILY.
17?
forma are rare and this may have some biochemical significance.
However, acyclic, bi-, tri-, tetra- and pentacy clic forms are
known in large number3.
Abietic acid (66) and all other diterpenes can hypothe
tically be derived from the C0 ̂ regular, head to tail polyiso-
prenoid or geranylgeranyl pyrophosphate units (7 l )0 Geranyl
22linalool (72) isolated from Jasmine oil is an allylic form of
(7l). Phytol (60) and Vitamin A ("3) are hydroderivatives of
2 o—2 4geranyl-geraniol . Gyclisation of geranyl-geraniol through
three isoprene units, folded as two potential chair cyclohexane
rings results in the formation of a molecule which by subsequent
oxidation of two substituents yields cateric acid^° (74)-(76)
(Scheme 4 o11). Ionization of terminal nucleophile through a
manool skeleton gives rise to pimarane skeleton of tricarboxylic
9 g 9 7diterpenes such as pimaradiene" ,w (79)0 A potential relation
ship between diterpenes with the pimarane and those with an
12abietane skeleton has been given by Ruzicka (Scheme 4012).
An interesting feature of a majority of the presently
known cyclic diterpenes is the absence of C-3 hydroxyl group and
presence oi the conventional 5o( ti0 p configuration of ring A/BOQ
as in steroids and triterpenes. Diterpenoid cassaine is an
exception with C-3 hydroxyl group. The generation of furan ring
in cafes tol (69) and kaliweoi (70) has been visualized as resul
ting from a vitagner-Meerwein rearrangement of hydxoxylated
precursors (Scheme 4„13)0
1 7 8
I 72 )
I 73)
1. - H
2- [0]
©
176)
s c h e m e :4 . i i
SCHEME 4.12. IONIC M ECHAN ISM IN THE B IOGENESIS OF
D IT ER P E N E S .
SCHEME.4.13.BIOSYNTHESIS OF FURAN RING OF
KAHWEOL.
180
The origin of diterpenoid nucleus has received its
greatest support from experiments from two mold products.
« 14Following the feeding of 2- C-mevalonic acid lactone to
29 30Trichothecium roseum L0, Birch and Arigoni independently
demonstrated that the diterpene rosenonolactone (9l) isolated
14 / vcontained the C pattern (Scheme 4 .14/. The biogenesis presu
mably proceeds through the hypothetical precursor (87) composed
of four isoprenoid units* Bosenonolactone (9l) itself does not
obey the classical isoprene rule in that it cannot be divided
into four regular isopentane units. Both the workers detected
the presence of radioactivity in the C-15 methyl group of ring
A and its absence in the carbon from the lactone ring. This
finding indicated that the C-15 was derived specifically from
the C-2 of mevalonic acid. The other mold product which has
proved to be of great value in biogenetic studies is gibberellic
acido Geranylgeranyl pyrophosphate formed from mevalonic acid
cyclises to a bicyclic intermediate (92) then to the tricyclic
diterpenoid (93)0 Gibberellic acid then arises by formation of
ring D from the side chain of (93), contraction of ring B with
extrusion of C-7 as the carboxyl group, followed by the loss of
angular methyl group and hydroxylation and formation of the
lactone group on ring A (Scheme 4015).
An interesting variation in diterpenoidal forms occurring
in nature is well illustrated by the structural formula for
31-32pleuromutilin (96) (Scheme 4.16).
1 8 3
14c 4 ^ T x CH2o H
©
H^ lV / cU
u (161
(8?
r T c u
I oh1
90)
SCHEMED,14 BIOGENESIS OF ROSENOLACTONE FROM
2 - C 14-M EV A LO N IC ACID LACTONE.
1 8 2
(93) ( 9 2 )
* =: C
SC H EM E.4.15. B IOSYNTHESIS OF G IB B E R E L L IN .
183
SCHEM E.4.16.BIOSYNTHESIS OF PLEUROM UTIL IN .
Diterpenoid quinones:
These quinones are simply oxidised terpenes<> The details
about the biogenesis of these quinones is not known. Tymoquinone
is the simplest example and the formation of thymol from mevalo-
33nate has been confirmed in Orthodon .iaponicmn (Labiatae). So far
helicobasidin, a fungal metabolite from Helicobasidium mompa is
the only terpenoid quinone whose origin from mevalonate has been
34 3 5established 0 Several pathways from farnesyl pyrophosphate
to helicobasidin are consistent with the observed labelling pattern.
Bisabolene hypothesis:
The involvement of Y -bisabolene (97), cuparenene (98),
cuparene (99) and deoxyhelicobasidin (lOO) was suggested by Natori
3 6et al. , without taking into account certain stereochemical conse
quences. The simplest cyclisation of trans-cis-farnesyl pyrophos
phate (lOl) yields trans bisabolene (102) and similarly cis-cis-
farnesyl pyrophosphate yields cis bisabolene (l03)o Moreover, by
further initiation of well recognised rearrangements, both bisa-
07bolenes may be derived from either isomer of farnesyl pyrophosphate .
If the further intermediates did not allow a randomization of the
labelled position in the six membered ring, mevalonate-2-^^G would
give rise to one of the labelled helicobasidins (104) or (105) or
to a mixture of both. However, the postulated intermediate,
cuparene would lead to randomization between the positions arbi
trarily numbered in (104) and (105) as 1 and 3 and between 4 and 5.
Another possible labelling pattern in helicobasidin is that repre
sented in (106) (Scheme 4.17).
184
1 8 5
Farneayl pyrophosphate
(98 )
(99)
I 101)
i'
I 100)
H 0 2 ) OT ( 103)
I( 98 )
1( 99)
I
( 106)
O H • == u c•f2 — half th#
sp. ac t ivi ty of •1105 )
S C H E M E . 4.17
IV.3 PRESENT WORK
1 8 6
The tricyclic diterpenoid quinones isolated in the present
study have quinonoid 1 O’ ring. This feature is biogenetically
very interestingo It is a well known fact that normal diterpenoids
arise from four units of mevalonate, and aromatic rings of other
secondary metabolites originate either from acetate or the aromatic
amino acids, namely, phenylalanine or tyrosine, Tbe quinonoid
moiety of other quinones have been biosyntbesized frota tyrosine
also.. Therefore, diterpenoid quinones would either originate
from mevalonate or from tyrosine. Consequently, if mevalonic
14acid-2- C is incorporated into diterpenoid quinones then the
radioactivity is expected to occur at one of the methyls of gem
dimethyl, isopropyl methyl,at C (l) and C (7) carbon atoms.
14Similarly, if L-tyrosine-U— C is incorporated then the activity
should be found in quinonoid moiety only.
The biosynthesis of 7oc-acetoxyroyleanone in Salvia
moorcraftiana plants was selected for the present study as it was
found to be the major compound present when experiments were
carried out in the month of Junea
1 4Isolation of 7oc-acetoxyroyleanone- C;
To investigate the proposed biogenetic scheme 4.18,
14 ' Qmevalonic acid-2- C (0.5 mci, sp. activity 1012 x 10 dpm/mM)
14and L-tyrosine-U- G (0„1 mci, sp. activity 261 mci/mM) were
administered separately to two sets of three to four month old
flowering plants by wick technique. During the administration of
1 8 7
M V A
H©
rx
i p p
OH
OPP
DMAPP
Ox i d .
KMnO/
H .( 110 )
OPP
S C H E M E D . 18 .P R O P O SE D B IO G EN ES IS AND
DEGRADATION OF 7 oC -
A C ET O X Y R O Y LEA N O N E.
L-tyrosine-U- C, carrier was used resulting in dilution of the
tracer. The plants were harvested after 8 days. Following the
method similar to the isolation of inactive 7oC-acetoxyroyleanone
(Ch. II , p , QJb ), the radiolabelled compound was isolated, puri
fied and assayed till constant activity from each set of plants
was obtained. It was observed that only one of these two precur-
14sors v iz., mevalonic acid-2- C was incorporated into 7oC-acetoxy-
royleanone. The yield of the compound, activity and its percen
tage incorporation is given in table 4 .1 .
14Table 4 01. Percentage incorporation of mevalonic acid-2- C
and the activity of undiluted 7oC-acetoxvroyleanone
isolatedo
14
Activity$
Precursor Yield
(n»g)
dpm/mg dpm/mM incorporation
14Mevalonic acid-2- C 60 217 0o8lxl0° 0 .001
To locate the position of radioactivity in the labelled
7oc-acetoxyroyleanone, degradative studies were carried ou on
this compound. The degradations were first standardised with
inactive 7cc-acetoxyroyleanone. The products obtained were chara
cterised by spectroscopic methods or by comparison with the
naturally isolated compoundSo The same degradative methods were
then applied to the radiolabelled compound.
1 8 9
The compound 7oc-acetoxyroyleanone on alkaline hydrolysis
yielded 70t~hydroxyroyleanone (108) which on dehydration with
p-toluenesulphonic acid in p-xylene afforded 6 ,7-dehydroroyleanone
(109)„ Oxidation of the latter compound with alkaline KMnO ̂
furnished a mixture of acids which on methylation with ethereal
diazomethane yielded a mixture of esters. However, the yield of
degraded products even in case of inactive sample was very low
thus making the task of isolating the desired acid ( l'lty from the
labelled 7<?c-acetoxyroyleanone extremely difficult. Therefore,
only the first two steps of degradation of the radiolabelled
compound were carried out» The activity found in these two steps
of reactions was found to be almost constant. This was sufficient
to establish that mevalonic acid is the overall precursor for
compounds having quinonoid 1C* ring.
It may be mentioned here that 7oc-acetoxyroyleanone could
be isolated as the major product from S. moorcraftiana collected
in the month of June. However, from plants collected in May, the
major compound isolated was 6,7-dehydroroyleanonea It appears
that the latter is the intermediate from which 7—acetoxy derivative
is formed via 6,7-epoxide, 7oC-hydroxy compound and followed by its
acetylation, which appears to be the last step in the biosynthesis.
Thus the low $ incorporation in 7oc-acetoxyroyleanone is accountable.
It is possible that if the biosynthetic experiments had been
carried out in the month of May, then 6 ,7—dehydroroyleanone would
have contained higher amount of radioactivity.
190
Chemicals: The following chemicals were used in the present
study: sodium sulphate (anhydrous, AR, Sarabhai), sodium hydro
xide (AR, Sarabhai), potassium permanganate (AR, E. Merck),
sodium bisulphite (LR, BDH), sodium bicarbonate (LB, BDH),
phosphoric acid (LB, BDH), hydrochloric acid (LB, BDH), BBOT
(Hewlett - Packard), and p-toluenesulphonic aci d (LR, BDH).
14 ,Badioactive chemicals: Mevalonic acid-2- C (0o50 mci, 209o4 mg,
9 14sp„ activity 1012 x 10 dpm/mM) and L-tyrosine-U- C (0ol mci,
0.07 mg, sp. activity 261 mci/mM) were obtained from Isotope
Division, Bhabha Atomic Besearch Centre, Trombay, Bombay-85.
Plant material: Fresh plants growing in Shankaracharya hills of
the Kashmir Valley were used for the isolation of diterpenoid
quinones. For the biosynthetic studies, these plants were
selected from the same region of the valley. Three to four-months
old plants were selected in the month of June for these experi
ments o
Methods:
Chromatography methods: Column and thin layer chromatography
were performed using silica gel adsorbent as per the procedure
described earlier (Chapter II, p.S'i ).
Physical methods: The instruments used for recording melting
points, UV, IB, NMB, Mass spectra have been specified earlier
(Chapter II, p. go ) 0
IV.4 EXPERIMENTAL
1 91Counting of radioactive samples: Badioactive samples were
counted on Packard Tri-Carb Liquid Scintillation Spectrometer.
The scintillation solution was prepared by dissolving BBOT :
2,5-bis/ 2-(5-tertbuty1 benzoxazoyl) thiophene / (4 g), in
, v 1 4toluene (AR/o Mevalonic acid-2- C was added as an internal
standard to correct for quenching. The sample to be counted was
dissolved in the minimum amount of a suitable solvent (benzene
or methanol, AR) and to this 10 ml of the scintillation solution
was added *
Isolation of 7ot-acetoxyroyleanone: 7°G-Acetoxyroyleanone was
isolated from Salvia moorcraftiana plants as per the procedure
already described in Chapter IIA, p.
14Administration of mevalonic acid-2- C and isolation of
14 1 4 /C-acetoxyroyleanone: Mevalonic acid-2- C (0.5 mci, 209*4 mg,
9sp. activity 1,12 x 10 dpm/mM) was dissolved in distilled water
(6 ml) and then administered to six flowering plantsQ A thread
was inserted into the stem of each plant (at about one third
height), the free ends of which were dipped in 1 ml of the precur
sor solution contained in a 5 ml beaker tied near the plant on a
wooden support* The solution was absorbed by the plant through
capillary action within a period of 6-8 hrs* After total take up
of the solution each beaker was carefully rinsed with more of
distilled water (0*5 - 1 ml) and the washings were allowed to be
absorbed. The process was repeated twice to ensure maximum admi
nistration of the precursor. The plants were harvested after
1 9 2
8 days and worked up for isolation of radiolabelled 7 -acetoxy-
royleanone by the usual methods
The plant material (200 g, dry weight) was extracted
with petroleum ether (60-80°) from which a concentrate (5.1 g)
was obtainedo This on chromatography using silica gel gave
7<t-acetoxyroyleanone (62 mg)6 It was purified by repeated crysta
llisation (methanol, m0p. 212°) to constant activity (60 mg, sp.
activity 0.81 x 10^ dpm/mM).
14 14Administration of L-tvrosine-U- C: L-Tyrosine-U- C in 0.01N HCl
solution (0.1 mci, o07 mg, sp. activity 261 mci/mM) was diluted
with the earrier compound (lO mg) and was made up to 3 ml with
0.01N HCl. The precursor solution was administered to three
plants by 'wick technique* as described above. The harvested
plants (110 g, dry weight) yielded peto ether concentrate (3«5 g).
This on column chromatography (silica gel) gave 7oc-acetoxyroylea-
none (42 mg). The repeated crystallization of the crude compound
yielded pure 7oc-acetoxyroyleanone (40 mg, m.p. 212°). The isolated
7cC-acetoxyroyleanone when assayed for radioactivity was found to
be totally inactive.
Degradation of 7QG-acetoxyroyleanone: The degradative studies were
carried out first with cold 7<x~acetoxyroyleanone and then with
the radiolabelled compound.
I • Conversion of 7oC-acetoxyroyleanoae into 7oC—hydroxyr oyleanone:
To the boiling solution of 70C-acetoxyroyleanone (53 mg)
in ethanol (3„5 ml) was added 0.10N NaOH (8 ml). The resulting
magenta coloured solution was refluxed gently for two hours, cooled
on an ice bath and acidified with 15$ phosphoric acid0 The solid
that separated out was extracted with chloroform and dried over
anhydrous sodium sulphate® After usual work up, the crude 7 -aceto-
xyroyleanone was purified by preparative TLG over silica gel,
followed by two crystallisations from aqueous methanol (20 mg, m.p<>
172°). It was found to be identical with the isolated natural
sample from this plant in all respects (UV, IB, NMR, MS, TLC, Co-TLC),
I I . Dehydration of 7<*-hydroxyroyleanone to 6,7-dehydroroyleanone;
A mixture of 70C-hydroxyroyleanone (20 mg), xylene (dry 1 ml)
and p-toluenesulphonic acid (0.5 rag) was refluxed for 3.5 hrs.
The reaction mixture was then concentrated under reduced pressure
and purified by preparative TLC followed by repeated crystallization
from methanol to yield pure red rectangular prisma of 6,7-debydro-
royleanone (14 mg, m.p® 171°). Spectral data (UV, lit, MS) was
found to be in agreement with natural 6,7-dehydroroyleanone isolated
from this plant (Chapter II, p0 q s ).
IIIo Alkaline potassium permanganate oxidation of 6.7-dehydroroyleanone
Aqueous potassium permanganate solution (4%) was added to
a solution of 6,7-dehydroroyleanone (l4 mg) in 2Jo aq. NaOH (2 ml)
at such a rate that an excess of potassium permanganate solution
was maintained in the reaction mixture. The temperature was main
tained at 40-45° initially for 2 hrs and then at 65-70° for 1 hr.
The solution was then cooled to room temperature and made acid to
congo red with dilute HC1„ The precipitated Mn02 was decomposed
191
with sodium bisulphite0 The volume of the solution was reduced
to 1 ml and then treated with a saturated solution of sodium
bicarbonate. This was then extracted with ether (4 x 5 ml) in
order to remove non-acidic impurities. The aqueous phase was
then acidified with dil» HG1. The precipitated acid was extracted
with ether (4 x 5 ml). The combined organic extracts were washed
once with brine and then dried over anhydrous sodium sulphate.
Removal of ether yielded a mixture of acids (2.5 mg).
IV. Ksfcerification of acid mixture;
The mixture (205 mg) was dissolved in ether and esterified
by treating with an excess of ethereal diazomethane <, Removal of
the solvent yielded a mixture of esters in extremely low amounts
and thus could not be separated either by prep„ TLC or prep. GLC.
195BIBLIOGRAPHY
lo Weiss, Uo and Edwards, J.M„, *The Biosynthesis of Aromatic
Compounds’ , John Wiley and Sons, New York and the references
cited therein, p., 144 (1980).
2„ Wallach, 0M Annalen 239, 1 (1887).
3. Buzicka, L ., Angew«, Chem0 51, 5 (l938)»
40 Sandermann, W„ and Schweers, W„, Tetrahedron Letters, J_,
257 (1962)0
50 Shaykin, S., Law, J 0, Phillips, A«H», Tchen, T.T. and
Bloch, K ., Proc. Nat. Acado Sci. (Wash„) 44, 998 (l958)o
60 Lynen, F ., Chem. Weekblo 43, 581 (l960)o
7 o Wolf, D .E ., Hoffman, G»H., Aldrich, P .E ., Skeggs, H .B .,
Wright, L.D. and Folkers, K„, Jo Amer. Chem. Soc0 78,
4499 (1956).
8o Clayton, U .S ., Quart. Rev. 1£, 168 (1965).
9o Nicholas, H0J . , In "Biogenesis of Natural Compounds"
(ed cBeraf eld, P0) 2nd Ed „ Pergamon Press (Oxford) p. 829(1967).
10. Birch, A»J., Boulter, D .E ., Fryer, B .I« , Thomson, PoJ.
and Willis, J .L ., Tetrahedron Letters, 1 (1959).
H o Popjak, G ., Tetrahedron Letters, 19 (1959).
120 Buzicka, L ., Experientia, 9, 357 (1953).
13. Mayer, B0, Z. Chem. 1_, 161 (l96l).
150
16.
17.
18.
19o
20 e
21.22.
23.
24.
25.
14. Richards, J.H. and Hendrickson, J .B ., in "The Biosynthesis
of Steroids, Terpenes and Acetogenins", W»A. Benzamin Inc0
(New York), p. 211 (l964)0
Growly, M.P., Godin, P = J ., Inglis, H .S . , Snarey, Mo and
Thain, E0M., Biochem. Biophyso Acta 60, 312 (l962)„
Clark, K .J ., Fray, G .I ., Jaeger, R0H. and Robinson, R«,
Tetrahedron 6, 217 (1959).
Hendrickson, J .B . , Tetrahedron, 7_, 82 (1959).
Parker, W., Roberts, J.S. and Homage, R ., Quart. Rev. 21,
331 (1967)o
Roberts, J .S ., "Chemistry of Terpenes and Terpenoids"
(Ed. Newman, A .A .) Academic Press (London), p 88 (1972)„
Herout, V ., In "Aspects of Terpenoid Chemistry and Bio
chemistry" (Edo Goodwen, ToW.) Academic Press (London),
p 63 (1971).
Rucker, G., AngeWo Chem. Internato Edit. 12, 793 (1973).
Lederer, E ., France Perfums, 3, 28 (i960).
Fischer, F .G ., Ann., 69. 464 (1928).
Karrer, P ., Morf, R. end Schopp, K ., Helv. Chim. Acta.
14, 1036, 1431 (1931).
Grant, F .W ., and Zeiss, H.H., J. Am. Chem. Soc. 7j5, 5001
(1954).
Briggs, L .H ., Cain, B.F. and Wilmshurst, J .K ., Chem. &
Ind. (London), 599 (1958).
19G
1 9 7
270 Ireland, R0E« and Schiess, P.W., Tetrahedron Letters,
37 (1960).
28o Djerassi, C., Cais, M. and Mitscher, L .A ., Jo Amer.
Chem. Soco 81, 2386 (1959)<,
29o Birch, A»J. and Smith, B ., In A Ciba Foundation Symposium
on the Biosynthesis of Terpenes and Sterols, p. 245, Jt> & A.
Churchill, Ltd„, London, 19590
30o Arigoni, D., ibid, p. 23l0
31e Richards, J .H ., and Hendrickson, J .B ., "Biosynthesis of
Terpenes, Steroids and Acetogenins, Frontiers in Chemistry
Series", W„Ao Benjamin, Inc0, New York, p , 255 (1964).
32o Birch, A«J«, Cameron, D.W. and Holzapeel, C.W., Chem. and
Indo (London), 374 (1963).
33o Yamazaki, Mo, Usui, T. and Shibata, S ., Chem. Pharmo Bull,
(Tokyo) 11, 363 (l963)»
34® Natori, S ., Inouye, Y. and Nishikawa, Ho, Chem. Pharm.
Bullo (Tokyo) 15, 380 (1967)o
35o Bentley, B. and Chen, Do, Plytochemistry 8, 2171 (1969).
36o Natori, S ., Nishikawa, H. and Ogawa, H ., Chem. Pham.
Bullo (Tokyo) 12, 236 (1964).
37o Buzicka, L®, In the Chemistry of Nato Products, Vol0 2,
p. 493, Butterworths, London (1963).