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INTRODUCTION TO SECONDARY PLANT

CONSTITUENTS - I
Dr. Harish & Purvi kakrani
TERPENOIDS

Terpenoids are the largest & structurally the most various class of naturally occuring secondary
plant metabolites, They are found normally in all plants (higher & lower) + in animals + in
insects, They are generally lipid soluble & are located in the cytoplasm of the plant cell, Most of
them occur free in the plant tissue but many are found as glycosides & esters of organic acids &
in some cases in combination with proteins, some organic acids are lipids + non-polar organic
solvents which are soluble with cinnamic or acetic acid which are the most common

Terpenoids are the most diverse chemical group naturally, some are acyclic & the others are
cyclic

Extraction & Detection


Terpenoids are normally extracted from plant tissues with petroleum ether, chloroform &
diethyl ether. They can be separated chromatographically on silica gel or alumina, The lower
members are volatile so that they can be obtained by distillation methods

All the terpenoids can be extracted by non-polar solvents


They are colourless (except carotenoids) & there is no sensitive specific chromato-reagent for
their detection, the common reagents used for their detection arevanillin\H2SO4, antimony
trichloride\HCL & anisaldehyde\H2SO4

TERPENOIDS FUNCTIONS
A number of quite different functions had been described such as the following
A) As growth regulators; sesqui-terpenoids (abscisins) are growth inhibitors while di-terpenoids
(gibberellins) are promotors
B) The coloured terpenoids (like carotenoids) are involved as an accessary pigments in the
process of photosynthesis
C) Some lower terpenoids provide plants with distinct smells (as I said before, the lower
members are volatile in nature like most of the chemical compounds of low organic formula) so
acting as insect attractants or repellant, used for defence
D) Some non-volatile terpenoids have been implicated as sex hormones among fungi, while
others act as agents for communication among insects like ants

So we can see the huge diversity in the functions of this important natural chemical class in the
biology of plants & animals in general

TERPENOIDS CHEMISTRY
Terpenoids are related by a common origin & a common structural relationship,so although they
have a wide diversity but still they are classed under the same origin chemically, They are
composed of multiples of a five carbon atom unit (or its isomer) known as ISOPRENE (iso-
pentenyl unit, prenyl, C5H8) for this, they are often referred as isoprenoid compounds, the unit
is shown in the following pictures
The unit is a C5H8 unit & is composed of a figure called (Head) which contains a =CH2 & a
methylene group (CH3) & a (TAIL) composed of only =CH2 & a normal hydrogen, resonance
may occur between the carbon atoms also due to the appearance of a 2 double bonds in the
isoprene structure
This simple 5 carbon unit is the responsible for the key reactions in terpenoidal biosynthesis, The
union of two C-5 residues in a head to tail manner with the formation of a C-C carbon gives rise
to the C-10 unit (geranyl pyrophosphate), Extension of this unit by the addition of a third unit
gives (farnesyl pyrophosphate) which is C-15 & continuation of the process yield C-20, C-25, C-
30, C-40…etc and the process can continue to give polyprenyl groups

We need to note that sometimes some compounds might be included also in the building process
which are not exactly isoprene units although they have similar shape and emperical formula, but
not identical, they also might have a head & tail phenomena like the dimethylallyl units

The building unit Isoprene does not occur as free in nature but it occurs as a pyrophosphate units,
so it’s called Isoprenyl pyrophosphate units IPPP orDimethylallyl pyrophosphate DMPP or
one of their derivatives & both of those units are the actual units responsible for the building of
terpenoids & their biosynthesis in general as just mentioned before, so a revision for the
multiplication process we see that for example the 2′nd unit formed would be composed of c-10,
linked in a head to tail manner with some exceptions some times, the head to tail manner of
linkage is called theIsoprene Rule & at the end if the length increases than the tendency to be
cycled (cyclization) will also increase naturally

Note also that when 2 simple C-5 pyrophosphate units are united, the reaction will yield for
example one simple C-10 geranyl pyrophosphate & the resulted structure will contain only one
pyrophosphate unit inside it, that means that the reaction will cleave one pyrophosphate from one
of the entered units and free it away

Classification of Terpenoids
They are classified according to the number of isoprene units involved in their formation
into the following
A) Hemi-terpenoids (or Hemiterpenes), C5H8, 1 unit
B) Mono-terpenoids, C10H16, 2 units
C) Sesqui-terpenoids, C15H24, 3 units
D) Di-terpenoids, C20H32, 4 units
E) Ses-terpenoids, C25H40, 5 units
F) Tri-terpenoids, C30H48, 6 units
G) Tetra-terpenoids (carotenoids), C40H64, 8 units
H) Poly-terpenoids, (C5H8)n, which is > 8 units

Note that the first one which is CH8, the Hemiterpenoid, doesn’t occur naturally in nature alone
& also note that the # of additional carbon atoms will not change, but still the hydrogen amount
is not considered to be added in a stable manners & this is a very important point so that not to
have a fast guess without studying the structure itself, so there is no direct imperical formula or
relation between the # of carbon atoms & the relative number of the hydrogen atoms, unlike
normally most of the basic organic compounds

HEMI-TERPENOIDS, C5H8
Compounds of this class as mentioned several times before are rarely encountered in nature as
stable, isolable metabolic end products, but they exist in living cells as highly active substances,
for example dimethylallyl pyrophosphate (DMPP) and Isopentenyl pyrophosphate IPPP, the
difference between both of them is the location of the 2 double bonds within the internal
structure of the simple isoprene unit itself
Note that dimethylallyl pyrophosphate might be also abreviated as DMAPP
See the following picture of a saturated resonance IPPP

The pysophosphate units are the responsible for forming the linkage between 2 isoprene units in
general
IPPP & DMPP are the key compounds used for the biosynthesis of terpenoidal compounds, they
are considered as the Building Blocks for Terpenoids in chemistry terms
MONO-TERPENOIDS, C10H16
They are derived from GPP (geranyl pyrophosphate) by different reactions (oxidation or
dehydration) that do not change the number of the carbon atoms, they appear free in
nature or combined with sugars & proteins & are classified into 4 major categories as the
following
A) Open chain monoterpenoids, acyclic
B) Cyclohexanes monoterpenoids, mono- & bicyclic
C) Cyclopentases, iridoids=monoterpene lactones
D) Irregular monoterpenoids

A) Open chain monoterpenoids, acyclic


These are liquids and comparatively few in nature for example citral & linalool

Check the following picture for citral which is a volatile liquid, extracted by distillation &
appears few in nature

Check the following picture for linalool which is also present in nature as a perfume and is a kind
of tertiary monoterpine alcohol
Note also that in both we can see that when the number of carbon increases from C-5 to C-10 the
tendency for cyclisation increases also
B) Cyclohexanes monoterpenoids, mono- & bicyclic
The majority of monocyclic monoterpenoids have a p-methane carbon skeleton, While the
bicyclic can conventionally be divided into 5 groups these are as the following
1) Carane
2) Thujane
3) Pinane
4) Fenchane
5) Camphane or called also Bornane

p-menthane structure as the picture below


Note that p-menthane is available in Mentha Piperita, the most important 2 forms are p-
menthol & menthone, as the following pictures shows them RESPECTIVELY

P-menthol
Note that para menthol includes an alcohol group on the para position of menthane

Menthone
Note that menthone contains a ketone group on the menthane structure

Carane structure as in the picture below

Carane is not a rigid structure, it is undergoing cyclization to become bicyclic, note also that all
the five types are bicyclic in the structure
Thujane structure as in the picture below

Pinane structure as in the picture below


Another more clear structure for pinane is as the following picture shows

Fenchane structure is as the following picture shows

Fenchane doesn’t follow the Terpenoid rule, the biosynthesis studies shows that it origionate
from the Isoprene way, so because of that only it is included in terpenoids
Camphane structure is as the following picture shows, note that this compound is available
in camphor

C) Cyclopentanes, iridoids = monoterpene lactones

This subgroup comprises of several hundreds of monoterpenes all with cyclopentane skeleton of
the iridane type

Iridane structure

Note that iridane is made up of a base skeleton of a pentane structure or as I can say a cyclo-
pentane structure, if this cyclo-pentane structure or bond was cleaved, the result will be called
a Modified Iridane, this compound is used normally for the biological activity & for
chemotaxonomy functions In most cases a hemiacetal bridge links positions 7 & 9 to form a
pyran ring condensed to the cyclopentane ring, While a hydroxyl group at position 7 is
conjugated to glucose molecule, for example loganin

Iridoids as just mentioned before are a very important compounds in the nature, they are
occuring ormally also in glycoside forms as just mentioned before
Loganin structure

Another form or picture for the Loganin structure as the picture below

Note that there is a glucose molecule attached & that there is a pyran ring formed during the
biosynthesis as shown in the above pictures

D) Irregular monoterpenoids

They arise from the following


1) Regular structures through degradative process, like for example in cryptone
2) Ten carbon atoms compounds but not following the isoprene rule, such as in chrysanthemic
acid & artemisia ketone

Chrysanthemic acid structure as the following


artemisia ketone as the following picture shows the structure

Note that they are both used as an insecticidal agents


Notes On The Biogenesis Of Essential Oils
Copyright ©Tony Burfield May 2005.

Many full-time aromatherapy college study courses now require a rudimentary knowledge of the biogenesis of
essential oils. A recent non-scientific survey of aromatherapy teaching materials by this author revealed a poor &
outdated comprehension of the subject (hence the generation of these notes and references).

Essential oils are the fragrant volatile products of the secondary metabolism of plants, and are generally are
composed of:

1. Volatile materials synthesised via the biogenic precursor isopentenyl pyrophophosphate (IPP), consisting of
complex mixtures of mono- and sesqui-terpene hydrocarbons, and (usually monofunctional) oxygenated materials
biogenically derived from them [i.e. put simply: terpenoids].

2. Phenyl propanoids from the shikimic acid pathway, and their corresponding biotransformation products [put simply:
many aromatics].

3. Other substances arising from the metabolism of fatty acids and amino acids, nitrogen & sulphur compounds, and
artefacts arising from the degradation of natural components during the isolation of essential oils from plant material,
and those arising during storage. Taking a few examples, aliphatic (unsaturated, non-terpenoid) undecatriene
hydrocarbons contribute to the odour of galbanum oil Ferula galbaniflua, and the heterocyclic compound indole
contributes importantly to the odour profiles of several floral oils and absolutes, including jasmine absolute
from Jasminum grandiflorum & J. sambac. The odour profile of Coriander oil Coriandrumsativum includes
contributions from substituted pyridines, thiazoles and pyrazines, such as 2,5 and 2,6-dimethyl pyrazines, whose
odour notes can be characterised as roasted, nutty and chocolate-like, with green undertones.Cis-3-hexenol with its´
intense green grass odour, a major component of green tea essential oil Camellia sinensis, and a minor component
of many herbaceous oils, together with other unsaturated C6 aldehydes, arises in green tissues whenever they are
cut or attacked by insects. It is produced by the enzymatic degradation of the linolenic acid produced from a branch of
the oxylipin pathway & catalyzed by the hydroperoxide lyase enzyme (Hatanaka et al.1987).

In many plants essential oil components are either found free, or bound up as glycosides, which are thought to play a
role in biogenesis of terpenoids as transport forms or as accumulation forms in undifferentiated cell cultures.

Older Theories of Terpenoid Biogenesis.

Isoprene Rule: Wallach (1887) proposed that monoterpenoids were hypothetically constructed by linkage of isoprene
units (in head to tail form).

(Head) (Tail)
Problems with this theory include the fact that satisfactory evidence of a biogenic route from isoprene, although it is a
natural product, has never been put forward. Secondly, the isoprene rule does not easily explain the formation of
irregular terpenoids, such as the tropolones found in oil of Western Red Cedar Thuja plicata, or some cyclopentane
iridoids (monoterpene lactones) – which Banthorpe (1994) describes as derivable from GPP by any simple cation
route [by now Jensen & Schripsema (2002) have described the biosynthesis of iridoid glycosides via iridodiol in the
Gentiaceae]. Similarly the biogenesis of some common irregular compounds, such as lavandulol in lavender oil, could
not be explained [now explained via the cleavage of a substituted cyclopropane compound formed by the
condensation of DMAPP with DMAPP].

Ruzicka´s Biogenic Isoprene Rule (1959): proposed that terpenes are built up from the biogenic 5-carbon compound
isoprene (2-methyl-1,3 butadiene).

This rule fitted in with the elucidation of the structures of camphor and alpha-pinene by head to tail addition of
biogenic isoprene units. The word turpentine, in which alpha-pinene occurs, gave rise to the compound class:
´terpene´. Ruzicka’s biogenic isoprene rule can now be taken to mean that sub-groups of terpenoids were built from a
single parent compound (permit the possibility of rearrangements during biosynthesis), and that parent compounds
were related in a homologous fashion (see table 1).

e.g. alpha-pinene, a bicycyclic monoterpene hydrocarbon based on a pinane structure, which has 10 carbon units
and can be built up from 2 the attachment of two isoprene units (represented by thick lines in diagrams below); and
the sesquiterpene b-caryophyllene, based on the caryophyllane 15 carbon skeleton, can be built from the attachment
of 3 isoprene units (see thick lines below):

Diagram 1: Building up Terpenoid Structures [after Pybus & Sell (1999)].


There are a large number of possible terpenoid skeletons which are outside the scope of this brief summary, but
which can be seen in publications such as that of Teisseire (1994).

More Modern theories : Mevalonic Acid Pathway to Terpenoids.

An enzymically modulated pathway route is envisaged to proceed via the formation of the 6-carbon compound 3R(+)-
mevalonic acid, thought as the precursor of all terpenoids.

Isopentenyl pyrophosphate (syn. 3-methylbutenyl pyrophosphate) can be regarded as the biogenic equivalent to
isoprene. e.g. for monoterpenoids:
Glucose -> Acetyl CoA (from citric acid cycle) -> Acetylacetyl CoA + Acetyl CoA -> 3-hydroxy-3-methylglutaryl CoA ->
mevalonic acid -> 5-phoshomevalonate -> 5 pyrophospho mevalonate -> IPP (isopentenyl pyrophosphate) + DMAPP
(dimethylallyl pyrophosphate) -> geranyl pyrophosphate (GPP) -> monoterpenes.

The formation of neryl pyrophosphate from GPP provides the carbocation A (see below) which is easily deprotonated
or rearranged to a wide range of cyclic, acylic or bicyclic skeletons. The skeletal terpene framework can be re-
arranged, oxidised, reduced, hydrated etc. to produce range of terpene products, according to the activity of
individual terpene cyclases. In higher plants, monoterpene synthesis in generally acknowledged to occur in plastids,
whereas sesquiterpene biosynthesis takes place in the cytosol (Kesselmeier & Staudt 1999).

++
->

(or)

Diagram 2. Showing GPP built up from IPP & DMAPP.

-> -> -> ->

Diagram 3. Production of alpha-pinene from GPP

GPP + IPP ->

Diagram 4. Showing FPP built up from GPP.

Sesquiterpenes are thus generated from farnesyl pyrophosphate; diterpenes from


geranylgeranyl pyrophosphate (see below).

Carbon No. Terpenoid class Parents ofExample Occurs in


sub-classes
C5 Hemiterpenoid IPP; DMAPPPrenol (syn. 3-Ylang-ylang
methyl-2- oilsCanangaodorata subsp.genuina
butenol), prenyl
esters
C10 Monoterpenoid GPP Limonene Citrus oils (Citrusspp.)
C15 Sesquiterpenoid FPP beta- Clove oilSyzigiumaromaticum
Caryophyllene
C20 Diterpenoid GGPP Sclareol Clary sage absolute Salvia sclarea
C25 Sesterterpenoid GFPP Ophiobolin A Certain

Fungi
C30 Triterpenoid Squalene Isophytol Jasmin absolute,
wax Jasminumgrandiflorum
C40 Carotenoid Phytoene beta-Carotene Green plant tissues

Table 1: Terpenoids, Precursors and their Natural Occurrence, modified from


Bancroft (1994).
[Key: IPP = isopentenyl pyrophophate; DMAPP = 3,3-dimethylallyl pyrophoshate; GPP= geranyl pyrophosphate; FPP
= 2E,6E-farnesyl pyrophosphate; GGPP = 2E, 6E, 10E-geranylgeranylpyrophosphate; GFPP = 2E,6E,10E-14E-
geranylfarnesyl pyrophoshate].

The Mevalonate Independent Pathway.

Criticisms of the mevalonate pathway have included the poor incorporation of radio-labelled 14C into carotenoids from
plant chloroplasts (Rohmner 1999). Recently a new pathway (a plastid-localised ‘mevalonate independent’ pathway
or ‘MEP pathway’) -> IPP, via the condensation of pyruvate and glyceraldehydes-3-phoshate to generate 1-deoxy-D-
xylulose 5-phosphate, followed by intramolecular rearrangement and reduction to 2-C-methylerythritol 4-phosphate,
has been discovered in higher plants and certain eubacteria including Escherichia coli. It is thought that the overall
formation of IPP in higher plants runs independently in these two pathways, the mevalonate pathway being confined
to the cystostolic compartment only. An article discussing the state of the art knowlege of the MEP pathway was
published by Rohmer (2003) available athttp://www.iupac.org/publications/pac/2003/pdf/7502x0375.pdf.
According to Kesselmeir & Staudt, the following pathway can be postulated:

Pyruvate -> Hydroxy ethyl thiamine pyrophosphate (+ 3-glycerylaldehyde-3-phoshate) -> 1-deoxy-D-xylulose 5-


phosphate -> IPP.

Thus the MEP pathway to IPP formation mechanism can potentially facilitate the formation of isoprene, carotenoids,
and other isoprenoids in higher plants (Schwender et al., 1996; Lichtenthaler et al., 1997; Zeidler et al., 1997).

Shikimic Acid Pathway.

Shikimic acid is formed from glucose in plants, and is the biogenic precursor of the amino acids L-phenylalanine, L-
tyrosine and L-typtophan. Pathways from shikimic acid generate anthranilates (e.g. in mandarin oil Citrus reticulata),
cinnamates (e.g. in peru balsam oil Myroxylon pereirae) and other phenylpropanoids, and from this point on to other
metabolites such as lignans & flavononoids. In particular, phenyl propanoids (basically compounds with a 3-carbon
chain attached to a benzene ring) are formed from trans or (E)-cinnamic acid via the elimination of ammonia from L-
phenylalanine. Common phenylpropanoids in essential oils include methyl chavicol, methyl eugenol, eugenol, methyl
cinnamate, vanillin & anethole.

Curiously not too much is known about certain aspects of phenyl propnaol accumulation in plants. An investigation of
accumulation of phenylpropenes in the two types of glandular trichomes (peltate & capitate) in two chemotypes of
Sweet Basil (eugenol & methyl chavicol) oil leaves (Gang 2001) revealed that the eugenol and methyl chavicol
accumulate almost exclusively in the peltate glands and the putative enzymes in the pathway are almost exclusively
confined to the peltate class. A simplified pathway for the production of methyl chavicol from phenylalanine was given
as follows:
-> -> -> ->

-> -> ->

References for further reading:

Banthorpe D.V. (1994) “Terpenes” In Mann J., Davidson RS., Hobbs J.B., Banthorpe D.V. & Harborne J.B. Natural
Products: Longman Scientific & Technical 1994.

Bu’Lock J.D. (1965) The Biosynthesis of Natural Products McGraw-Hill.

Croteau R. (1981) in Biosynthesis of Isoprenoid Compounds, (eds J.W. Porter et al.), Wiley, New York, Vol. 1, p. 225.

Croteau, R. (1987) “Biosynthesis and Catabolism of Monoterpenoids” Chem. Rev., 87, 929.

Croteau, R. et al. (1994), Recent Adv. Phytochem., 28, 193.

Gang D.R. et al. (2001) “An Investigation of the Storage and Biosynthesis of Phenylpropenes in Sweet Basil” Plant
Physiol 125, 539-555.

Hatanaka A., Kajiwara T., Sekiya J. (1987) “Biosynthesis pathway for C6-aldehydes formation from linolenic acid in
green leaves.” Chem Phys Lipids 44, 341–361.

Hill, R.A. (1993) in The Chemistry of Natural Products, 2nd edn (ed. R.H. Thomson), Blackie, Glasgow, pp. 107.

Jensen S.R. & Schripsema J. (2002) “Chemotaxonomy and pharmacology of Gentianaceae” In Gentianaceae:
Systematics & Natural History eds Struwe L. & Albert V. Cambridge Univ. Press 2002.

Kesselmeier J. & Staudt M. (1999) “Biogenic Volatile Organic Compounds (VOC): An Overview on Emission,
Physiology and Ecology” Journal of Atmospheric Chemistry 33: 23–88, 1999.

Lichtenthaler H. K., Schwender L., Disch A. & Rohmer M. (1997) “Biosynthesis of isoprenoids in higher plant
chloroplasts proceeds via a mevalonate independent pathway” FEBS Lett. 400, 271–274.

Pybus D.H. & Sell C.S. The Chemistry of Fragrances RSC 1999.
Rohmer M. (1999). In Comprehensive Natural Product Chemistry. Isoprenoids Including Carotenoids and Steroids, D.
E. Cane (Ed.), Vol. 2, pp. 45–67, Pergamon, Oxford, UK (1999) and references therein (through Romer 2003).

Schwender J., Seemann M., Lichtenthaler H. & Rohmer M. (1996) “Biosynthesis of isoprenoids, carotenoids, sterols,
prenyl sidechains of chlorophylls and plastoquinone) via a novel pyruvate/glyceraldehydes 3-phosphate non-
mevalonate pathway in the green alga Scenedesmus, Biochem. J. 316, 73–80.

Teisseire P.J. (1994) Chemistry of Fragrant Substances VCH Publishers Inc.

Zeidler J. G., Lichtenthaler H. K., May H. U. & Lichtenthaler, F.W. (1997) “Is isoprene emitted by plants synthesized
via the novel isopentenyl diphosphate pathway?” Z. Naturforsch. 52c, 15–23.