natural product - جامعة نزوى · pdf filenatural product. bacteria and fungi ......

The organic compounds isolated from the living organism

i.e plants, animals and micro organism are generally known

as Natural products. These includes carbohydrates, protein,

aminoacids, alkaloides, terpenes, antibiotics etc

The term natural product is applied to material derived from

plants microorganisms and invertebrates, which are fine

biochemical’s factories for synthesis of both primary and

secondary metabolites

Natural Product

Bacteria and Fungi as source for

Biologically active Compounds

Classical and Advance methods

Structure of Morphine

O

HO

H

NH

CH3

HO1

2

3

4

5

6

7

8

9

10

11

12

1314

15 16

Morphine (Astramorph)

HO- Group is needed for activity

HO- Group not important to activity

‘Tinkering’ with the structure of

morphine produced heroin

O

HO

H

NH

CH3

HO1

2

3

4

5

6

7

8

9

10

11

12

1314

15 16 O

AcO

H

NH

CH3

AcO1

2

3

4

5

6

7

8

9

10

11

12

1314

15 16

Morphine (Astramorph)Heroin (Diamorphine)(2X as potent as morphine)(Conversion of two -OH groups to -OAcfacilitates crossing of the BBB)

Easily enzymatically hydrolyzed to AcOH and HO-ArHO- Group is needed for activity

HO- Group not important to activity

The heart beat may be too fast or too slow

Terpenoids

INTRODUCTION

Terpenoids are the secondary metabolites synthesized by plants,

marine organisms and fungi by head to tail joining of isoprene

units. They are also found to occur in rocks, fossils and animal

kingdom.

Isoprene (short for isotepene), or 2-methyl-1,3-butadiene, is a

common organic compound with the formula CH2=C(CH3)CH=CH2.

Under standered conditions it is a colorless liquid. However, this

compound is highly volatile because of its low boiling point.

Isoprene Isoprene

Terpenes

•Terpenes are natural products that

are structurally related to isoprene.

H2C C

CH3

CH CH2

or

Isoprene

(2-methyl-1,3-butadiene)

Isoprene

Head

Tail

Head

Tail

Isoprene

Head

Tail

Head

Tail

Isoprene

• Terpenes and terpenoids posses a carbon fram work

consisting of five carbon units known as isoprene unit.

• It is represented by symbol C5H8

• In oldest days the term terpene was used for those

compounds containing 10 carbon atoms.

• This is still used in Modern classification of Terpenes.

Terpenes are classified in to the following groups.

Hemeterpens 1 x C5H8 = C5H8

Monoterpens 2 x C5H8 = C10H16

sesquiterpens 3 x C5H8 = C15H24

Diterpens 4 x C5H8 = C20H32

sesterpens 5 x C5H8 = C25H40

triterpens 6 x C5H8 = C30H48

Tetraterpenes 7 x C5H8 = C35H58

Polyterpenes n x C5H8 = C5H8)n

Rubber n= 100 or above

CALASSFICATION

TYPE OF NUMBER OF ISOPRENE

TERPENOIDS CARBON ATOMS UNITS

hemiterpene

monoterpenoid

sesquiterpenoid

diterpenoid

triterpenoid

tetraterpenoid

C5

C10

C15

C20

C30

C40

one

two

three

four

six

eight

hemi = half di = two

sesqui = one and a half tri = three

tetra = four

NOTE

:

sesterterpenoid C25 five

Mnonoterpenoids

Monoterpenes are a class of terpenes that consist of

two isoprene units and have the molecular formula

C10H16.

Monoterpenes may be linear (acyclic) or

contain rings. Biochemical modifications

such as oxidation or rearrange-ment

produce the related monoterpenoids.

Representative Monoterpenes

a-Phellandrene

(eucalyptus)

Menthol

(peppermint)

Citral

(lemon grass)

O

H

OH

Representative Monoterpenes

a-Phellandrene

(eucalyptus)

Menthol

(peppermint)

Citral

(lemon grass)

O

H

OH

Representative Monoterpenes

a-Phellandrene

(eucalyptus)

Menthol

(peppermint)

Citral

(lemon grass)

Mnonoterpenoids

Acyclic monoterpenoid:

¦Â-myrcene

CH2OH

nerol

CHO

geranial

Monocyclic monoterpenodi

OH

l-menthol menthone

O

cineole

O

Bicyclic monoterpenoid

¦Á-pinene

d-borneol

OH

Acyclic monoterpenoid

Biosynthetically, isopentenyl pyrophosphate and dimethylallyl pyrophosphate are

combined to form geranyl pyrophosphate

Geranyl pyrophosphate

Acyclic monoterpenoid

Elimination of the pyrophosphate group leads to

the formation of acyclic monoterpenes such as

ocimene and the myrcenes.

Myrcene

Acyclic monoterpenoid

Hydrolysis of the phosphate groups leads

to the prototypical acyclic monoterpenoid

geraniol.

geraniol

Acyclic monoterpenoid

Additional rearrangements and oxidations

provide compounds such as citral,

citronellol, and many others.

citral

Acyclic monoterpenoid

Many monoterpenes found in marine

organisms are halogenated, such as halomon.

Halomon is a polyhalogenated monote

rpene first isolated from the marine

red algae Portieria hornemannii.

It has attracted research interest becau

se of its promising profile of selective cy

totoxicity that suggests its potential

use as an antitumor agent.

Halomon

Monocyclic monoterpenoid

In addition to linear attachments, the isoprene

units can make connections to form rings. The

most common ring size in monoterpenes is a

six-membered ring.

A classic example is the cyclization of

geranyl pyrophosphate to form limonene.

Bicyclic monoterpenoid

Geranyl pyrophosphate can also undergo two

sequential cyclization reactions to form bicyclic

monoterpenes, such as pinene which is the

primary constituent of pine resin.

Bicyclic monoterpenoid

Other bicyclic monoterpenes include carene

and camphene.

Camphor, borneol and eucalyptol are examples

of bicyclic monoterpenoids containing ketone,

alcohol, and ether functional groups,

respectively.

carene

camphor borneol

Sesquiterpenoids

– Sesquiterpenes are a class of terpenes that consist of

three isoprene units and have the molecular formula

C15H24.

– Like monoterpenes, sesquiterpenes may be acyclic

or contain rings, including many unique combinations.

Biochemical modifications such as

oxidation or rearrangement produce the

related sesquiterpenoids.

Representative Sesquiterpenes

a-Selinene

(celery)

H

Representative Sesquiterpenes

a-Selinene

(celery)

H

Representative Sesquiterpenes

a-Selinene

(celery)

Sesquiterpenoids

• Acyclic sesquiterpenoids

• Monocyclic

sesquiterpenoids

¦Á-farnesene ¦Â-farnesene

O

• Bicyclic sesquiterpenoids

OH

¦Á-eudesmol

H

H

cadinene

guaiazulene

Acyclic Sesquiterpenoids

When geranyl pyrophosphate reacts with

isopentenyl pyro- phosphate, the result is the 15-

carbon farnesyl pyrophosphate, which is an

intermediate in the biosynthesis of sesquiterpenes

such as farnesene . Oxidation can then provide

sesquiterpenoids such as farnesol.

Sesquiterpenes are found naturally in plants as

defensive agents.

farnesyl pyrophosphate

farnesene

farnesol

Monocyclic Sesquiterpenoids With the increased chain length and additional

double bond, the number of possible ways that

cyclization can occur is also increased, and there

exists a wide variety of cyclic sesquiterpenes. In

addition to common six-membered ring systems such

as is found in zingiberene, a consitituent of the oil

from ginger, cyclization of one end of the chain to the

other end can lead to macrocyclic, rings such as

humulene.

姜稀zingiberene

Bicyclic Sesquiterpenoids

In addition to common six-membered rings

such as in the cadinenes, one classic bicyclic

sesquiterpene is caryophyllene, from the oil of

cloves which has a nine-membered ring and

cyclobutane ring. Additional unsaturation provides

aromatic bicyclic sesquiter- penoids such as

guaiazulene.

guaiazulene

H

H

cadinene

caryophyllene

Tricyclic Sesquiterpenoids

With the addition of a third ring, the possible

structures become increasingly varied. Examples

include longifolene, copaene and the alcohol

patchoulol.

longifolene copaene isomers of patchoulol

Diterpenoids

Diterpenoids

Diterpenes are composed for four isoprene units and have

the molecular formula C20H32. They derive from

geranylgeranyl pyrophosphate.

Examples of diterpenes are cafestol, kahweol, cembrene

and taxadiene (precursor of taxol).

geranylgeranyl pyrophosphate

cafestol cembrene

Representative Diterpenes

Vitamin A

OH

Representative Diterpenes

Vitamin A

OH

Representative Diterpenes

Vitamin A

Diterpenoids

Diterpenes also form the basis for biologically important

compounds such as retinol, retinal, and phytol. They are

known to be antimicrobial and antiinflammatory. The herb

Sideritis contains diterpenes.

retinol retinal

Structure ----Diterpenoids

Acyclic diterpenoids

CH2OH

phytol

Bicyclic diterpenoids

O

OHO

CH2OH

HO

andrographolide

Monocyclic diterpenoids

CH2OH

vitamin A

COOH

pimaric acid

Tetracyclic diterpenoids

H

H

H

kaurene

Tricyclic diterpenoids

Triterpenoids

Triterpenoids

Triterpenes consist of six isoprene units and have the

molecular formula C30H48. The linear triterpene squalene, the

major constituent of shark liver oil, is derived from t he

reductive coupling of two molecules of farnesyl

pyrophosphate. Squalene is then processed biosynthetically

to generate either lanosterol or cycloartenol, the structural

precursors to all the steroids.

squalene

Farnesyl pyrophosphate

lanosterol cycloartenol

Representative Triterpene

Squalene

(shark liver oil)

tail-to-tail linkage of isoprene units

O-P-O-P-O

O O

O O

O-P-O-P-O

O O

O OO-P-O-P-O

O O

O O

O-P-O-P-O

O O

O O

O-P-O-P-O

O O

O O

GPP

FPP

O-P-O-P-O

O O

O OFPP

O-P-O-P-O

O O

O OFPP

Squalene

Squalene

α-carotene

β-carotene

• Acyclic triterpenids squalene

• Bicyclic triterpenids

Structure ----Triterpenids

Structure ----Triterpenids

• Tetracyclic triterpenids Dammarane Lanostane

Tirucallane

H

H

H

dammarane

H

H

H

H

lanostane H

tirucallane

H

H

Cycloartane Cucurbitane

cycloartane

H

cucurbitane

H

H

H

H

• Pentacyclic triterpenoids

Oleanane Ursane Lupane

oleanane

H

H

H

H

ursane

H

H

Hlupane

H

H

H

H

Structure ----Triterpenids

It is the most important member of the

most of the acyclic monoterpenoides.

Because the structure of most of the

other compounds in this group are

based on that of citral. Its is widely

distributed and occurs to an extent of

60-80% in lemon grass oil. It is a liquid

which has the smell of lemons.

Citral C10H16O

The molecular formula of citral is C10H16O

analogue of saturated hydrocarbons

CnH2n+2

C10H10x2+2 = C10H22

IHD: 22-16/2 = 3

From IHD it is found that the citral contains 3 double bonds

Structure of Citral.

On cat. Hydrogenation 2 moles of hydrgone

were consumed. This showed that the

molecule contain 2 C=C bonds

Cat. Hydrogentation

CHO

Cat. Hydrogenation

CHO

2 moles

CHO

OHC

2 moles

Br2

Br

Br

Br

Br

On bromination also two molecule of bromine were consumed.

This also proved that molecule contain 2 C=C bonds.

Citral was converted into an oxime on treatment with NH2-

OH. This reaction showed that molecule contains an oxo

group.

CHO

2 moles

H2N=OH

CH=N-OH

• Citral is reduced to an alcohol geraniol (

C10H18O) . Geraniol is a primary alcohol and the

formation a primary alcohol form carbonyl

compound confirmed that the citral contains an

aldehyde functional group.

CHO

Na/Hg

CH2-OH

geraniol

• Oxidation of citral with Ag2O gives geranic

acid C10H16O2 and no loss of carbon atom

takes place. This further proves that oxo

group in citral is an aldehyde group.

CHO

Ag2O

CO2H

geranic acid

• Reaction with potassium hydrogen sulphate

• On heating with KHSO4 citral form P-Cymene.

This reaction was used by semmler to determine

the position of methyl and isopropyl group in the

skeleton structure 1

CHO

KHSO4

P.cymene1

This reaction showed

that the citral

molecule is acyclic in

which two isoprene

Units are joined in

head to tail manner

• The examination of the formula of citral

shows that 2 geometrical isomers are

possible

• The functional group may be Cis or Trans.

CHO

(a)

H

CHO

(b)

H

TransCis

These structure are

supported by NMR

spectroscopy

• Synthesis of citral can be carried out by

the following synthetic route.

Br

Br

HC

COMe

COMe

Na

Br

CH

COMe

COMeO Zn/I-CH2-CO2Et

H

Reformatsky Rtn

OH

CO2Et

AC2O

Co2Et

Ca salt

(HCO2)Ca

CHO

NaOH

-H2O

CHO

Cat. Hydrogenation

CHO

2 moles

C10H16O2Br2

C10H16Br4O

CHO

2 moles

H2N=OH

CH=N-OH

• Citral is reduced to an alcohol geraniol (

C10H18O) . Geraniol is a primary alcohol

and the formation a primary alcohol form

carbonyl compound confirmed that the

citral contains an aldehyde functional

group. CHO

Na/Hg

CH2-OH

geraniol

Essential Oils

Essential Oils

• Terpenes and terpenoids are the primary constituents of the essential oils of many types of plants and flowers.

• Essential oils are used widely as natural flavor additives for food, as fragrances in perfumery, in aroma therapy, and in traditional and alternative medicines. Synthetic variations and derivatives of natural terpenes and terpenoids also greatly expand the variety of aromas used in perfumery and flavors used in food additives.

ISOLATION & SEPARATION

TECHNIQUES

Essential oils containing mono- and

sesquiterpenoids are obtained by water and or

steam distillation of the part such as flowers,

leaves or stems, where the essential oils occur

in more concentrated form. Due to the heat

lability of certain constituents of essential oils

different distillation methods have to be used

for different raw materials which are briefly

described below:

Distillation

• Today, most common essential oils, such as lavender,

peppermint, and eucalyptus, are distilled.

• Raw plant material, consisting of the flowers, leaves,

wood, bark, roots, seeds, or peel, is put into a

n alem bic (distillation apparatus) over water.

• As the water is heated the steam passes through the pla

nt material, vaporizing the volatile compounds. The vap

ors flow through a coil where they condense back to

liquid, which is then collected in the receiving ve

ssel.

Distillation

Most oils are distilled in a single process. One except

ion is Ylang- ylang (Cananga odorata), which takes 2

2 hours to complete through a fractional distillation.

Distillation

The recondensed water is referred to as a hydrosol, herbal di

stillate or plant water essence, which may be sold as another fra

grant product.

Popular hydrosols are rose water, lavender water, lemon balm, a

nd orange blossom water.

The use of herbal distillates in cosmetics is increasing.

Some plant hydrosols have unpleasant smells and are therefore

not sold.

Expression

Most citrus peel oils are expressed mechanically, or cold-pressed.

Due to the large quantities of oil in citrus peel and the relatively low

cost to grow and harvest the raw materials, citrus-fruit oils are cheaper

than most other essential oils. Lemon or sweet orange oils that are

obtained as by-products of the citrus industry are even cheaper.

Prior to the discovery of distillation, all essential oils were extracted

by pressing.

Solvent extraction

• Most flowers contain too little volatile oil to undergo

expression and their chemical components are too delicate

and easily denatured by the high heat used in steam

distillation. Instead, a solvent such as hexane or

supercritical carbon dioxide is used to extract the oils

• Extracts from hexane and other hydrophobic solvent are

called concretes, which is a mixture of essential oil, waxes,

resins, and other lipophilic (oil soluble) plant material.

Solvent extraction

• Although highly fragrant, concretes contain large

quantities of non-fragrant waxes and resins. As such

another solvent, often ethyl alcohol, which only

dissolves the fragrant low-molecular weight

compounds, is used to extract the fragrant oil from the

concrete

• The alcohol is removed by a second distillation, leaving behind

the absolute

ISOLATION & SEPARATION

TECHNIQUES

• Terpenoids • Following methods are employed for the extraction of mono-,

sesqui-, di-, tri-, and tetraterpenoids.

Air dried powdered part of the plant is extracted by percolation

or soxhlet extraction successively with organic solvents with

increasing polarity such as petroleum ether, benzene, diethyl

ether, chloroform, ethyl acetate, acetone, ethanol, methanol

and water. The extraction efficiency can be increased with the

decrease in the time of the process by stirring the pulverized

plant material using mechanical stirrer with the chosen solvent

and filtering it to obtain the extract.

STRUCTURE ELUCIDATION

• Physical Mehtods 1. Molecular formula

2. Specific rotation

3. Refractive index

• Spectral Methods for Structure

Determination 1. UV

2. IR

3. MS

4. NMR

Physical Mehtods 1. Molecular formula Determination of the molecular formula of an isolated pure

terpenoid is done by finding out the empirical formula and

molecular weight. Empirical formula can be found out by

elemental analysis .While molecular weight can be

determined by vapour density, elevation of boiling point

and depression of freezing point.

2. Specific rotation Specific rotation of a compound is measured to ascertain the

optical activity exhibited by it. It helps to distinguish between

optical isomers.

3. Refractive index It is measured to calculate the value of molecular refraction,

which is useful to find out the nature of the carbon skeleton

especially in the case of sesquiterpenoids .

Spectral Methods

1. UV Functional groups, present in terpenoids , which

absorb in the UV range between 200-350nm are termed as chromophores.However UV data becomes valuable only when the terpenoid molecule contains conjugated double bonds and/or α,β-unsaturated carbonyl group.

2. IR

This method is routinely used for the identification as well as the structure elucidation of new terpenoids.

3. MS

FAB-MS affords the e xact molecular ion peak along with diagnostic fragmentation patterns of the terpenoid molecule. It is an important tool for the structure determination .

Spectral Methods 4. NMR

• NMR spectroscopy comprising of both PMR and CMR is in fact one of the

Most important tools furnishing a good teal of information required for the

structure elucidation.

The combination of 1D selective and 2D NMR techniques such as COSY,

TOCSY, ROESY,2D IN-ADEQUATE, HMQC, HMBC COLOC, HOHAHA,

HETCOR and selective INEPT are of great value for the structure elucidation

of various terpenoids including the saponins and glyosides of a

number of sugar moieties.

EXAMPLE

• C10H16O

• b.p.77℃

• UV:236nm

• IR:1665，1625，1603，1398，1190，1117cm-1.

• MS:m/z 69(100),41,84,94,109,67,83,81

• 1H-NMR:1.65(6H，d， C-7 methyls) ，2.15(3H，s，C-3 Me)，5.0(1H，t， H-6)， 5.8(1H，d，H-2)， 9.84(1H，d，H-1).

• 13C-NMR:190(C-1)，127.5(C-2)，162.1(C-3)，40.5(C-4)，26.5(C-5)，123.5(C-6)，132.3(C-7)，25.3(C-8)，17.4(C-9)，17.0(C-10).

CHO

geranial