BIOL 158:

BIOLOGICAL CHEMISTRY II

Lecture 9:

TERPENES

Lecturer:

Christopher Larbie, PhD

• The volatile component of fragrant materials in plant

belong to a class of compounds called terpenes.

• Terpenes/terpenoid compounds are widely distributed in

nature mostly in plants. They are polymers of isoprene

units joined head to tail.

• An isoprene unit contains 5-carbon atom put together in a

regular pattern, usually head-to-tail in terpenes up to 25

carbons

• The terpenes have been long associated with the term

Essential Oils comprising resins, steroids and rubber

• In fact, they are hydrocarbons that usually contain one or

more C=C double bonds, while the terpenoids are

oxygen-containing analogues of the terpenes

• They are thoroughly distributed in the plant kingdom,

especially in those plants that have abundant chlorophyll

2-Methyl-1,3-butadiene

• The double bond in the isoprene units are in the trans

geometric configuration. Isoprene units are produced from

acetyl CoA or acetic acid

• Terpenes can be cyclic or open chain molecules, carbon-

carbon saturated or unsaturated and may contain a

variety of functional groups such as aldehydes, alcohols,

hetons and esters

• Terpenes occur in rubber, carotenoids in carrots,

potatoes, yellow vegetables, vitamin A, E and K,

chlorophyll and urbiquinone.

• Terpenes containing 30 carbons or more are usually

formed by the fusion of two smaller terpene precursors

such that the head-to-tail "rule" appears to be violated

• In overall, terpenes hold potential interest practical

applications especially in the fragrance and flavour

industries, as well as in the pharmaceutical and chemical

industries.

Classification of Terpenes

• No of C No of Isoprene units Class of terpenes

• 5 1 Hemeterpene

• 10 2 monoterpenes

• 15 3 sesquiterpenes

• 20 4 diterpenes

• 25 5 sesterterpenes

• 30 6 triterpenes eg. Steroids

• 40 8 tetraterpenes eg. Carotenoids

• ˃40 78 polyterpenes eg. Rubber

Methods of extraction

1. Expression; squeezing the juice out of plant material

2. Steam distillation; suitable for the lower members

especially up to 20 carbon compounds.

3. Extraction with volatile solvent; vacuum distillation and

chromatographic method.

Isoprene Rule

• A compound is said to be a terpenoid if it can be broken

into 5-carbon units usually joined head to tail

• Example

Examples of Terpenes

• Monoterpenes are naturally occurring compounds, the

majority being unsaturated hydrocarbons (C10)

• But, some of their oxygenated derivatives such as

alcohols, ketones, and carboxylic acids are known as

monoterpenoids

• The branched-chain C10 hydrocarbons comprises of two

isoprene units and is widely distributed in nature with

more than 400 naturally occurring monoterpenes

identified

• Moreover, besides being linear derivatives (Geraniol, Citronellol), the monoterpenes can be cyclic molecules (Menthol – monocyclic; Camphor – bicyclic; Pinenes (α and β) – Pine genera) as well

• Thujone (a monoterpene) is the toxic agent found in Artemisia absinthium (wormwood) from which the liqueur, absinthe, is made

• Borneol and camphor are two common monoterpenes. Borneol, derived from pine oil, is used as a disinfectant and deodorant

• Camphor is used as a counterirritant, anaesthetic, expectorant, and antipruritic, among many other uses

Sesquiterpenes • Sesquiterpenes are biogenetically derived from farensyl

pyrophosphate and in structure may be linear, monocyclic

or bicyclic

• They constitute a very large group of secondary

metabolites, some having been shown to be stress

compounds formed as a result of disease or injury

• One group is the Sesquiterpene Lactones. Over 500

compounds of this group are known; they are particularly

characteristics of the Compositae but do occur

sporadically in other families

• Not only have they proved to be of interest from chemical

and chemotaxonomic viewpoints, but also possess many

antitumor, anti-leukaemic, cytotoxic and antimicrobial

activities

• They can be responsible for skin allergies in humans and

they can also act as insect feeding deterrents.

• Hemigossypol and related aldehydes together with the

dimeric gossypol are sesquiterpenes stress compounds

found in the subepidermal glands, immature flower buds

and seed kernels of the cotton plant (Gossypium spp.)

• Besides having insecticidal properties, gossypol is of

considerable pharmaceutical interest in that in humans, it

functions as a male anti-infertility agent

• In China, gossypol has been used to treat infertility in

males and studies have shown an increase in sperm

production

Diterpenes:

• The diterpenes, which contain 20 carbons (four C5 units), include

phytol (the hydrophobic side chain of chlorophyll), the gibberellin

hormones, the resin acids of conifer and legume species,

phytoalexins, and a host of pharmacologically important

metabolites, including taxol, an anticancer agent found at very low

concentrations (0.01% dry weight) in yew bark, and forskolin, a

compound used to treat glaucoma

• Some gibberellins have only 19 carbon atoms and are considered

norditerpenoids since they have lost 1 carbon through a metabolic

cleavage reaction

• Vitamin A, which combines with protein opsin to form the active

pigment of the retina

Triterpenes • These contain 30 carbon atoms, are generated by the

head-to head joining of two C15 chains, each of which constitutes three isoprene units joined head-to-tail

• This large class of molecules includes the brassinosteroids, the phytosterol membrane components, certain phytoalexins, various toxins and feeding deterrents, and components of surface waxes, such as oleanolic acid of grapes

• Squalene in shark liver oil, smaller quantities are in olive oil, rice bran oil, yeast, wheat oil. Squalene is used in biosynthesis of steroids

OH

H

-amyrin in resin(cyclic)

CH3

CH3

CH3

CH3 CH3

CH3 CH3 CH3

Squalene (acyclic)

Tetraterpenes: • The most prevalent tetraterpenes (40 carbons, eight

isoprene units) are the carotenoid accessory pigments which perform essential functions in photosynthesis.

•Polyterpenes:

• These contain more than eight isoprene units, include the prenylated quinone electron carriers (plastoquinone and ubiquinone), long-chain polyprenols involved in sugar transfer reactions (e.g., dolichol), and enormously long polymers such as rubber (average molecular mass greater than 106 Da), often found in latex

• Natural rubber may be regarded as l, 4 adolition polymer, it is treated with elemental sulphur in a process called vulcanization

Biosynthesis of Terpenoid compounds

• The biosynthesis of all terpenoids can be divided into 4

steps

1. Synthesis of the fundamental precursor, Isopentenyl

pyrophosphate (IPP)

2. Repetitive addition of IPP to form a series of

prenylpyrophosphate homologs, which serve as the

immediate precursors of the different classes of

terpenoids.

3. Elevation of these allylic prenylpyrophosphates by

specific terpenoid synthases to yield terpenoid

skeletons.

4. Secondary modification of the skeletons (largely redox

reactions) to give rise to the functional properties and

great chemical diversity of this family of natural products

Compartmentation

• A more fundamental, and perhaps universal, feature of

the organization of terpenoid metabolism exists at the

subcellular level.

1. The sesquiterpenes (C15), triterpenes (C30), and

polyterpenes appear to be produced in the cytosolic and

endoplasmic reticulum (ER) compartments.

2. Isoprene, the monoterpenes (C10), diterpenes (C20),

tetraterpenes (C40), and certain prenylated quinones

originate largely, if not exclusively, in the plastids

• The evidence now indicates that the biosynthetic

pathways for the formation of the fundamental precursor

IPP differ markedly in these compartments, with the

classical acetate/mevalonate pathway being active in the

cytosol and ER and the glyceraldehyde

phosphate/pyruvate pathway operating in the plastids

The acetate/mevalonate pathway

1. Hydroxymethylglutaryl-coenzyme A (HMG-CoA) is the

precursor for terpenoid synthesis

• HMG-CoA is also an intermediate on the pathway for

synthesis of ketone bodies from acetyl-CoA. The enzymes

for ketone body production are located in the

mitochondrial matrix

• HMG-CoA destined for terpenoid synthesis is made

by equivalent, but different, enzymes in the cytosol. HMG-

CoA is formed by condensation of acetyl-CoA and

acetoacetyl-CoA, catalyzed by HMG-CoA Synthase

2. HMG-CoA reductase catalyzes production of mevalonate from HMG-CoA

• The carboxyl group of hydroxymethylglutarate that is in ester linkage to the thiol of coenzyme A is reduced first to an aldehyde and then to an alcohol

• NADPH serves as reductant in the 2-step reaction. Mevaldehyde is thought to be an active site intermediate, following the first reduction and release of CoA. HMG-CoA reductase is an integral protein of endoplasmic reticulum membranes

• The catalytic domain of this enzyme remains active following cleavage from the transmembrane portion of the enzyme

3. Mevalonate is phosphorylated by 2 sequential

phosphate transfers from ATP, yielding the

pyrophosphate derivative

Glyceraldehyde phosphate/pyruvate

pathway

• In plastids, pyruvate reacts with TPP to yiel a 2-C

fragment, hydroxylmethyl-TPP which condenses with

glyceraldehyde 3-phosphate

Synthesis of Terpenoids 1. Pyrophosphomevolanate decarboxylase catalyzes

ATP-dependent decarboxylation, with dehydration, to

yield isopentenyl pyrophosphate

• Isopentenyl pyrophosphate isomerase inter-converts

isopentenyl pyrophosphate

and dimethylallyl pyrophosphate

• The mechanism involves protonation followed by

deprotonation

2. Prenyl transferase catalyzes a series of head-to-tail

condensation reactions

• Dimethylallyl pyrophosphate reacts with isopentenyl

pyrophosphate to form geranyl pyrophosphate

• Condensation with another isopentenyl pyrophosphate

yields farnesyl pyrophosphate. Each condensation

reaction is thought to involve a

reactive carbocation formed as PPi is eliminated

• Prenyl transferase (Farnesyl Pyrophosphate Synthase)

has been crystallized with the substrate geranyl

pyrophosphate bound at the active site

2. Squalene synthase catalyzes head-to-head

condensation of 2 molecules of farnesyl pyrophosphate,

with reduction by NADPH, to yields qualene

• Tracer studies using 14C have confirmed that terpenoid compounds are all formed initially through acetate mevalonate pathway

• It has been shown that steroids are modified terponoid compound of cholesterol C27 may be regarded as C30 –C3. Carotenoids which are C40 may be regarded as terpenoid compounds

• Role of IPP

• To prevent esterification of the coo-funcctional group and makes the ester a good leaving group.

•

Carotenes • The term carotene (also carotin, from the Latin carota, or

carrot) is used for several related unsaturated

hydrocarbon substances having the formula C40Hx, which

are synthesized by plants but cannot be made by animals

• Carotene is an orange photosynthetic pigment important

for photosynthesis

• Carotenes are all coloured to the human eye. They are

responsible for the orange colour of the carrot, for which

this class of chemicals is named, and for the colours of

many other fruits and vegetables (for example, sweet

potatoes, chanterelle and orange cantaloupe melon)

• Carotenes are also responsible for the orange (but not all of the yellow) colours in dry foliage

• They also (in lower concentrations) impart the yellow coloration to milk-fat and butter

• Omnivorous animal species which are relatively poor converters of coloured dietary carotenoids to colourless retinoids have yellowed-coloured body fat, as a result of the carotenoid retention from the vegetable portion of their diet

• The typical yellow-coloured fat of humans and chickens is a result of fat storage of carotenes from their diets

• Carotenes contribute to photosynthesis by transmitting the light energy they absorb to chlorophyll

• They also protect plant tissues by helping to absorb the energy from singlet oxygen, an excited form of the oxygen molecule O2 which is formed during photosynthesis

• β-Carotene is composed of two retinyl groups, and is broken down in the mucosa of the human small intestine by β-carotene 15,15'-monooxygenase to retinal, a form of vitamin A

• β-Carotene can be stored in the liver and body fat and converted to retinal as needed, thus making it a form of vitamin A for humans and some other mammals

• The carotenes α-carotene and γ-carotene, due to their single retinyl group (β-ionone ring), also have some vitamin A activity (though less than β-carotene), as does the xanthophyll carotenoid β-cryptoxanthin.

• All other carotenoids, including lycopene, have no beta-ring and thus no vitamin A activity (although they may have antioxidant activity and thus biological activity in other ways)

α-carotene

γ-carotene

• All carotenes absorb in the UV/VIS region; it means that

UV can be used to quantitate carotenes in natural

compounds

Retinol • Carotenoids are tetraterpenes, widely distributed in both

plants and animals. They are called lipodromic pigment meaning fat soluble

• They are coloured, yellow violet orange or red. In higher plants, carotenes are found in the leaves and chlorophyll

• They constitute the principal pigments of certain yellows, orange and red flowers

• Types of Carotenoids

• Carotenes - hydrocarbons soluble in pet ether.

• Xanthophylls - oxygenated derivatives of the hydrocarbons of

STEROIDS • Steroids are compounds when distilled in the presence of

selenium give a hydrocarbon called cyclopentano perhydro phenanthrene ring

Classification of Steroids

• Sterols

• Bile acids

• Sex hormones

• Adrenal cortex hormones

• Steroidal glycosides

• Hydrocarbons

Sterols • These are steroids with alcoholic OH functional group.

Sterols are widely distributed in nature

• They may occur as free compounds or as esters of some

fatty acids e.g. cholesterol – most abundant of sterols.

Cholesterol in the precursor of all steroids in animals.

Cholesterol is a white crystalline solid

• Features of sterols

1. It has fused rings labeled ABCD (present in all steroids).

2. It has 2 angular methyl groups at position 10 and 13; the methyl carbons are C19 and C18.

3. Side chain is bonded to carbon 17. There are 2 configurations, cis and trans

• A/B junction for all naturally occurring steroids could be cis or trans while B/C acid C/D are always trans

• In natural cholesterol, C19 is up (above the plane of the ring).

• Functions of cholesterol

1. Component of cell membranes

2. Synthesis of bile acids

3. Antheroclerosis

4. Synthesis of sex hormones

5. Vit D synthesis

Biosynthesis of cholesterol

• Cholesterol is synthesized from isoprene units origin from

isopentenyl pyrophosphate.

• Squalene oxidocyclase catalyzes a series of electron

shifts, initiated by protonation of the epoxide, resulting

in cyclization

• Structural studies of a related bacterial enzyme have

confirmed that the substrate binds at the active site in

a conformation that permits cyclization with only modest

changes in position as the reaction proceeds

• The product of the cyclization reaction is the

sterol lanosterol

• Conversion of lanosterol to cholesterol involves 19

reactions, catalyzed by enzymes associated with

endoplasmic reticulum membranes

• Additional modifications yield the various steroid

hormones or vitamin D

• Many of the reactions involved in converting lanosterol to cholesterol and other steroids are catalyzed by members of the cytochrome P450 enzyme superfamily

• The human genome encodes 57 members of the cytochrome P450 superfamily, with tissue-specific expression and intracellular localization highly regulated.

• Some P450 enzymes are localized in mitochondria. Others are associated with endoplasmic reticulum membranes

• Cytochrome P450 enzymes catalyze various oxidative reactions

• Many are mixed function oxidations (mono-oxygenations),

that require O2 as well as a reductant such as NADPH

• One oxygen atom is incorporated into a substrate and the

other oxygen atom is reduced to water

• An example is hydroxylation of a steroid, as in the

endoplasmic reticulum electron transfer pathway depicted

at right, NADPH transfers 2 electrons to cytochrome

P450 via a reductase that has FAD and FMN prosthetic

groups.

• P450 substrates include steroids, polyunsaturated fatty

acids, eicosanoids, retinoids, and various non-polar

xenobiotics (drugs and other foreign compounds)

• Some P450 enzymes have broad substrate specificity.

Mechanisms for detoxification of non-polar compounds

include reactions such as hydroxylations that increase

polarity, so that the products of these reactions can be

excreted by the kidneys

Chemistry of steroids 1. Steroids undergo reactions characteristic of the

functional groups presents

2. Reactions are greatly influenced by stereochemistry

and steric factors-the fused ring presents a rigid

structure.

3. There are 2 angular methyls on the B face. Therefore

many reagents preferentially attack at the relatively

unhindered and face especially when the reactions take

place near the angular methyls

Reactions

a) Hydrogenation occurs across the double bond. It is

possible for the Hs to attack at the α-face because the

angular methyl in on the B face

b) Esterification

OH

H2/Pt

OH

HH

RCOOH

OHOH

O

OHOH

Nu

Bile acids • These are isolated from the bile of higher animals. They

occur as salts or amides of either glycine or taurine

• This makes the molecule partly polar and partly non polar.

Bile acids are synthesized from cholesterol

• Bile acids resemble cholesterol, but have more polar

constituents

• Like the fatty acids, which have polar and non-polar

portions that allow them to act like detergents, the bile

acids also act like detergents

• They are stored in the bile in the gall bladder and act to

solubilize dietary lipids during digestion

• Glycocholate is a major bile salt

• Bile salts provide one way to break down cholesterol in

the body and, as noted in the previous paragraph,

blocking their normal reabsorption in the intestine requires

the body to break more cholesterol down for their

synthesis

• Their functions include

1. They are emulsifying agent in the gut, acids indigestion

of liquids

2. They provide suitable pH for action of lipases in the

small intestine

3. They are metabolites of cholesterol

4. They promote transport of liquids through aqueous

medium (blood and body fluids)

• Positions of OHs are 3, 6, 7, 11, 12 and 23. In all natural bile acids have 3 OHs.

Vitamin D Synthesis • Vitamin D is derived from a cholesterol derivative, 7-

dehydrocholesterol, in a reaction that is initiated by ultraviolet light and which splits one of the rings

• The active form, calcitrol, is formed from Vitamin D3 by hydroxylation reactions in the liver and kidney

• Vitamin D deficiency causes a syndrome known as rickets, which is characterized by inadequate calcification of cartilage and bone

• Vitamin D deficiency in children was signficant in 17th century England. Today, many foods, including milk are fortified with vitamin D

HORMONES • Hormones are substances or chemical messengers produced

by endocrine glands that elicit specific effects in target tissues or organs

• They are required in minute quantities and are short-lived. E.g. insulin, glucagon, sex hormones, adrenocortical hormones, gastrointestinal hormones etc.

• An example of a plant hormone is ethylene (ethane). It regulates plant growth and fruit ripening and controls sex of flowers of certain plants

• 3-indole acetic acid in a naturally occurring hormone in plants that catalyzes fruit development, stem elongation, ethylene stimulation and synthesis, begins root formation in cuttings, decreases fruit loss and spoilage when the fruit drops from the tree

Steroid hormones

• The most important steroid hormones in vertebrates are

listed below

• Calcitriol (vitamin D hormone) is also included in this

group, although it has a modified steroid structure. The

most important steroid hormone in invertebrates is

ecdysone.

• Progesterone is a female sexual steroid belonging to the

progestin (gestagen) family. It is synthesized in the corpus

luteum of the ovaries

• The blood level of progesterone varies with the menstrual

cycle. The hormone prepares the uterus for a possible

pregnancy

• Following fertilization, the placenta also starts to

synthesize progesterone in order to maintain the pregnant

state.

• The development of the mammary glands is also

stimulated by progesterone.

• Estradiol is the most important of the estrogens. Like

progesterone, it is synthesized by the ovaries and, during

pregnancy, by the placenta as well

• Estradiol controls the menstrual cycle. It promotes

proliferation of the uterinemucosa, and is also responsible

for the development of the female secondary sexual

characteristics (breast, fat distribution, etc.).

• Testosterone is the most important of the male sexual

steroids (androgens)

• It is synthesized in the Leydig intersitial cells of the testes,

and controls the development and functioning of the male

gonads

• It also determines secondary sexual characteristics in

men (muscles, hair, etc.).

• Cortisol, the most important glucocorticoid, is

synthesized by the adrenal cortex

• It is involved in regulating protein and carbohydrate

metabolism by promoting protein degradation and the

conversion of amino acids into glucose

• As a result, the blood glucose level rises. Synthetic

glucocorticoids (e. g., dexamethasone) are used in drugs

due to their anti-inflammatory and immunosuppressant

effects.

•

• Aldosterone, a mineralocorticoid, is also synthesized in

the adrenal gland. In the kidneys, it promotes Na+

resorption by inducing Na+/K+ ATPase and Na+ channels.

• At the same time, it leads to increased K+ excretion. In

this way, aldosterone indirectly increases blood pressure.

• Calcitriol is a derivative of vitamin D. On exposure to

ultraviolet light, a precursor of the hormone can also arise

in the skin

• Calcitriol itself is synthesized in the kidneys. Calcitriol

promotes the resorption of calcium in the intestine and

increases the Ca2+ level in the blood.

Iodothyronines

• The thyroid hormone thyroxine (tetraiodothyronine, T4) and its active form triiodothyronine (T3) are derived from the amino acid tyrosine

• The iodine atoms at positions 3 and 5 of the two phenol rings are characteristic of them

• Post-translational synthesis of thyroxine takes place in the thyroid gland from tyrosine residues of the protein thyroglobulin, from which it is proteolytically cleaved before being released

• Iodothyronines are the only organic molecules in the animal organism that contain iodine. They increase the basal metabolic rate, partly by regulating mitochondrial ATP synthesis. In addition, they promote embryonic development.

PHEROMONES • Many animals and insects use chemicals to communicate

with others of the same species

• These chemicals are called pheromones. Pheromones

are substances secreted by insects or animals that trigger

a reaction in a member of the same species

• The reaction triggered may elicit

1. Immediate behavioural effect e.g. aggregation and

2. Primer effect, not immediate, physiological effect

• Types of Pheromones

1. Sex pheromones - this is species specific, it makes

sure they find a mate

2. Aggregation pheromones - helps to exert complete

attack on food or prey

3. Epideitic pheromones - helps to prevent

overcrowding; chemical mediated spacing of attack.

4. Alarm pheromones - helps to raise alarm of danger

to ants

• Sex Pheromones characteristics

1. Airborne

2. Contact

• They are perceived by antennae which have sense cells.

• Pheromones act on the sensory nerve cells at the mouth

parts or antennae

• Applications of pheromones

1. Monitoring population. Sex pheromone can be used to

lure and trap pests or insects. We can set up a trap and

put in it sex pheromone of a particular insect.

2. Pheromones can be used to cause disruption of mating

3. Pheromones can be combined with conventional

insecticides to control insect infestation of agricultural

fields.