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).